1.1. Chemicals
The Mafenide hydrochloride used in this work was purchased from TCI chemicals India. HPLC grade Methanol (Batch no-6962441221) was purchased from Qualigens Pvt. Ltd. India. Potassium dihydrogen phosphate was purchased from Finar Chemical Pvt ltd. (India).
1.2. Instruments
The experiments were conducted using Waters HPLC, model 2695 alliance equipped with a quaternary pump, solvent management system, degasser, 100µL capacity injector loop and a multiple wavelength Waters 2998 Photodiode Array (PDA) Detector. All the operations of the HPLC-PDA system were controlled by the Empower 3 software (Water Corporation, MA). All weighing was carried out on Sartorius CPA2250 calibrated analytical balance, which has a precision of 0.01 mg. All samples were prepared using ultrapure water from ELGA Lab Water (Evoqua Water Technologies LLC, Pittsburgh, PA).
1.2.1. Liquid chromatographic conditions
A 4.6 x150 mm, 5µm Inert sustain C8 column was loaded with 10µL test samples and flow rate of 0.8 mL/min was used for final separation with KH2PO4: Methanol (85:15 V/V as mobile phase). For most all the experiments, the column temperature was kept at 25°C. Before the operation, the mobile phase was filtered with a 0.45µm nylon filter and degassed in an ultrasonic bath. The analysis was done on λmax 222nm after scanning standard drug solution with a PDA detector (Fig. 2). All sets of analysis were performed at ambient temperature.
1.3. Preparation of mobile phase, standard, calibration curve and samples
1.3.1. Preparation of mobile phase
To prepare mobile phase A, 1.36 gm of 10 mM KH2PO4 was dissolved in 1000 mL of Ultrapure water. Before usage, the mobile phase was filtered using a nylon 0.45µm filter membrane. Combination of mobile phase was considered as 85:15 V/V (10mM KH2PO4: Methanol).
1.3.2. Preparation of the Standard solution 20ppm
A standard solution of MAF was prepared by dissolving 4 mg of MAF in 10 mL of methanol. From the above solution, the final concentration of 20 ppm was prepared by diluting 0.5 mL of standard solution to 10 mL of methanol.
1.4. Validation of HPLC method
Following the ICHQ2(R1) Guideline, we validated the HPLC method beginning with the specificity parameter and moving on to the range of analysis like linearity, precision, accuracy, LOQ, LOD, and robustness for formulation. All required parameters were tested according to ICH(Borman and Eler, 2017).
1.4.1. Specificity
Specificity is defined as the ability of a compound to be separated individually without interference from a placebo, excipient, or other impurities. Here, we injected a 20ppm concentration of the formulation to test its specificity.
1.4.2. System suitability
Testing for system appropriateness was crucial to the HPLC system and chromatographic conditions. To obtain accurate, precise, and repeatable results, we must guarantee system and chromatographic conditions before any study begins. The standard solution was injected six times to assess the system's applicability, and chromatographic parameters such as the tailing factor and theoretical plate were analysed. The system is appropriate if the tailing factor is less than 2, the resolution is greater than 2, and the theoretical plate is more significant than 2000.
1.4.3. Linearity and range
The range is a concentration matrix; any analysis performed within the range should be give reproducible and nearby results. For linearity, individual 5 different concentration solutions were prepared from the stock solution. The stock solution was prepared by dissolving 5mg in 50mL of volumetric flask using a diluent as methanol. Pipette out 0.5, 1, 1.5, 2.0, and 2.5 mL of the stock solution in 10 mL of volumetric flask respectively, to create 5, 10, 15, 20, and 25 ppm final solution. Each solution injected duplicate the calibration curve and linear regression equation were built by plotting the peak area at 222 nm against concentration.
1.4.4. Accuracy
The analytical process for determining how closely an experimental value corresponds to the actual amount of substance in a matrix is called accuracy. It is expressed as a percentage of the recovery. To evaluate accuracy, three different concentrations were prepared by standard addition methods a known amount of API spike in spray formulation solution to prepare 80%, 100%, and 120% solution (low, medium, high). All three solution (36ppm, 40ppm,44ppm) was injected in triplicate and recorded the response area. The results was calculated by using mentioned formula [14].
The acceptance criteria for accuracy were 90–110%.
% Recovery= (Recovered conc/injected conc.) x 100.
1.4.5. Precision
Reproducibility, intermediate precision, and repeatability are the three categories of precision listed in the ICH guidelines. Six replicates of the reference solution were injected to test the repeatability. Intermediate precision performed on different days, i.e., intra-day and inter-day. In present study solution of concentration of 10ppm for repeatability and 20ppm for intra and inter-day precision were injected, and response area and retention time recorded. Acceptance criteria for precision was ≤ 2% RSD.
1.4.6. Limit of quantification and limit of detection.
Limit of detection is lowest concentration of drug which can be detected but it not necessary to quantify. Methods for detection of LOD is by injecting lowest concentration till we can find small peak which is visible to eye called as visual evaluation methods, second on is based on Signal to noise ratio and last one calculated by using standard deviation of response and slope. In present study we used slope of calibration and standard deviation parameter to calculate the LOD. LOD = 3.3 X Standard deviation /Slope. Limit of quantification is the lowest concentration of drug were precision and accuracy should be acceptable limits. For calculation of LOQ = 10 X Standard deviation /slope.
1.4.7. Robustness
The robustness parameter was tested to ensured that developed method was is enough stable for any deliberated changes in system parameter like flow rate, temperature, wavelength changes withing specific limits and also for HPLC methods parameter like mobile phase composition, buffer pH, organic composition. As in industry, product life cycle is depending on market requirement so as per market demands batches will be plans for production and mean while quality will be checked. Most of analytical methods are HPLC, so it’s necessary to reproduce the same validated methods for any periods of time whenever its required and hence every time is difficult to maintain exact system and chromatographic condition to be match. That why we performed the robustness to ensured method is enough robust and maintain to produce acceptable and accurate result. This test performed by changing mobile phase composition, flow rate, and temperature. Assay value and retention time parameter were considered for evaluation.
1.4.8. Analysis of formulation
Assay of formulation
Content of formulation calculated by weighing 100 mg of spray solution, which is equal to 4 mg of drug and dissolved in 10 mL of methanol as diluent. filter the solution with 0.2µm nylon filter. From above solution 0.5mL again diluted with methanol up to 10mL to get 20ppm concentration. Injected in HPLC system.
For Actuation assay – formulation containing spray bottle tested for his drug delivery. Press one time spray bottle in small conical flask containing small amount of diluent methanol. Make to final volume up to 10mL in volumetric flask with methanol. From above solution 0.5mL further diluted with 10 methanol. Inject above solution in HPLC system to check contain.
1.4.9. Drug and Excipient interaction study
To study pre-formulation aspect before finalization of dosage form, it is necessary to know in advance possible interaction between excipient and drug. formulation will be stable if excipient do not show any interaction with drug physically and chemically. Study was design by taking 1:1 ratio of each excipient with drug, Isopropyl alcohol, Polyethylene glycol, Polypropylene glycol, Eudragit, Hydroxy propyl methyl cellulose in Eppendorf tubes. For liquid excipient agitation stress was used, all mixture was agitated for 1 hr. and keep under stability chamber 40°C/75%RH for 30 days. In case of solid excipient small amount of water(10µl) added and kept under stability chamber for 1 month 40°C/75%RH ([15]). All sample observed for physical changes like color and precipitate, for checking drug stability toward excipient, sample was prepared using methanol as diluent. filter with 0.45µm nylon filter and injected in assay method. Interaction results were monitored, refer Table 1 for chromatographic condition. Results was checked for absence of any interaction or any unknown peak and for physical appearance.
Table 1
HPLC Chromatographic condition.
Parameters | HPLC Assay method | |
---|
Mobile phase A | 10mM KH2PO4 | |
Mobile phase B | Methanol | |
Column | Inert sustain C8, C8, (4.6 x 250 mm, 5µm) | |
Flow rate | 0.8mL/min | |
Separation mode | Isocratic mode Mobile phase A: Mobile phase B 85:15V/V | |
Detection wavelength | 222nm | |
Injection volume | 10µL | |
Temperature | Ambient | |