A sort of validated Bioanalytical method developed for the estimation of Etoposide and Cisplatin in rat plasma by using two different advanced liquid chromatographic techniques like HPLC and UPLC and its application in bioequivalence studies

An easy, quick, precise, active and reproducible reverse method high performance liquid chromatography (RP-HPLC), as well as Ultra pressure liquid chromatography (UPLC) unique technique, was developed for the bioanalytical method of Etoposide and Cisplatin with Oxaplatin as an internal standard. Chromatographic analysis was performed using HPLC was Waters Alliance e-2695, and UPLCwasWaters Acquity UPLC by using x-bridge phenyl 150x4.6mm, 3.5μ column and therefore themobile phase containing 0.1% triethylamine and acetonitrile at a ratio out of 60:40 v/v. The low rate was 1 ml/min, and analytes were detected at 283 nm employing a photodiode array detector at ambient temperature in both liquid chromatographic systems. The proposed biological method was proved in both HPLC and UPLC with USFDA guidelines. USP tailing is 1.1, 1.02 for etoposide and 1.16, 1.05 for Cisplatin in HPLC and UPLC. USP plate count is 4794, 3884 for Etoposide and 9289, 14487 for Cisplatin. The calibration curve under accumulation set of 5-100 ng/ml of etoposide and 10-200 ng/ml of Cisplatin in both HPLC and UPLC. The accuracy of low, middle and high-level quality control samples are taken in 50%, 100% and 150% levels. Stability study was administered altogether conditions are bench top, wet extract and auto sampler, freeze-thaw, short term stability and in long term stability. This is the advanced technique established to the straightforward, economical, suitable, precise, accurate and stablemethod for the analysis of Etoposide and Cisplatin and study of its stability and pharmacokinetic studies using rat plasma.


INTRODUCTION
Etoposide is marketed as Etopophos and others may be physiotherapy (Alfarouk et al., 2015) medication used to treat the diverse group of cancer including seminoma (Ferri, 2017), carcinoma (O'Reilly et al., 2007;Garcia et al., 2016), lymphoma (Yang et al., 2015), leukaemia (Pasmant et al., 2009), neuroblastoma (Maris et al., 2007) and cancer of the ovarian (Ebell et al., 2016). It is often seen in hemophagocytic lymphohistiocytosis (Machowicz et al., 2016;Yildiz et al., 2020). It is used either by mouth or by injection into a vein. Adverse effects are very normal, including low blood levels, vomiting, end of existence (Langhans, 2000), diarrhoea (Smalley et al., 2019), loss of hair and fever. Some important side effects shall have allergies and poor vital sign. Usage during breast feeding is likely to damage the foetus. Etoposide is a drug family regulator of topoisomerase (AD'yakonov et al., 2017). It is suspected to be detected by destroying the DNA (Kumar et al., 2018;Carell et al., 2018). It is on the planets list of important drugs, the safest and strongest drugs required in the health care system. Etoposide has employed as well as a brand of cancer chemotherapy like carcinoma of Kaposi (Schneider and Dittmer, 2017). Ewing's sarcoma, carcinoma, seminoma, lymphoma, non lymphocytic leukaemia and glioblastoma multiforme. It is also offered along with other medication. It is often seen in a strengthening routine before bone marrow or blood somatic cell transplantation (Hande, 1998). Cisplatin may be a chemotherapy medication wont to treat a variety of cancers like ovarian, seminoma, cervical carcinoma, bladder, head and neck (Beyzadeoglu et al., 2014), oesophagal cancer (Sultan et al., 2016), carcinoma, mesothelioma (Kondola et al., 2016) brain and neuroblastoma these are all type of cancers. It is given by injection into a vein. General reactions of suppression of bone marrow, auditory issues, disorders and vomiting (Oun et al., 2018). Such signi icant results in numbness (Moon, 2014), walking issues, asthma, electrolyte issues and cardiac diseases. Using during breastfeeding, it is dangerous to the infant. Cisplatin is an antineoplastic class drug dependent on platinum. Cisplatin is an intravenous application of regular saline medication for strong and haematological malignancies, they are not advised management different forms of cancer, including sarcoma (DeVita et al., 2015), certain carcinomas (Halford, 2005) (small cell carcinoma, epithelial cell carcinoma at the top and ovarian tumors), lymphomas, bladder cancer (van Osch et al., 2016), cervical cancer (Bosch and de Sanjosé, 2007) and reproductive cell tumors (Omata et al., 2017). Cisplatin is highly powerful against seminoma. The chemical type of Etoposide and Cisplatin structures are in Figure 1 A and B.

Chemicals and reagents
Sun Pharmaceuticals, Mumbai kindly supplied etoposide and cisplatin.
Oxaliplatin has been used as an internal standard, obtained from Sigma-Aldrich, Hyderabad. Methanol, acetonitrile, orthophosphoric acid and triethylamine of HPLC grade were purchased from Rankem, Hyderabad and Ultrapure water was provided by the Milli Q water apparatus from Millipore Mumbai.  Chromatographic research was carried out administered by using it waters alliance e-2695 of HPLC and waters acquity model of UPLC data handling with Empower software, with similar conditions are xbridge phenyl 150x4.6 mm, 3.5 µ column. Therefore, the mobile phase containing 0.1% TEA and acetonitrile mixed in a proportion of 60:40 pm v/van isocratic elusion was applied on a river run about 1 ml/min. The sample was injected at volume 10 µl, and wavelength detection is 283 nm for both compounds in HPLC and UPLC.

Diluent
The mobile phase was used as diluents.

Control sample and internal control preparation of the study Preparation norm
Etoposide and Cisplatin normal stock strategies (300 and 600 ng/ml) was prepared by using diluents. Internal control stock of oxaliplatin was prepared in a concentration of the (1200 ng/ml) these control solutions has been made placed at below 4 • C and 4 • C guarded for light except for the internal standard functioning a remedy that was prepared every day.

Preparation of samples
For the preparation of a sample of 100 µl of plasma, 500 µl of acetonitrile, 500 µl of control stock and 500 µl of the internal control stock to precipitate all the proteins and blend within the vortex cyclo mixture. Centrifuge at 2000 rpm for 30 min. Collect the supernatant solution and ilter through 0.45 µ syringe ilter and collect the solution into a sample vial and run into the chromatographic systems.

Speci icity and Selectivity
Selectivity was based on performed by analyzing test the blueprint rat plasma from six nonidentical rats to check the instruction of retention time of analytes.

System precision
System precision was performed MQC level of standard solution is injected into six times to record the analyte area to calculate the %RSD was not exceed 2.0%.

Linearity
Linearity for the method was studied in two chromatographic techniques determined at concentrations ranging from 5-100 ng/ml and cisplatin 10-200 ng/ml.

LOD and LOQ
In the analyte, LOD is low-level concentration, that can be measured with an appropriate degree of accuracy and precision (signal to noise > 3.0) by the proposed method. LOQ is the lowest plasma concentration with a reasonable degree of precision and accuracy (signal to noise level >20). Etoposide and Cisplatin LOD and LOQ concentrations were 0.5 ng/

Impact matrix
Comparison of the height ratio inside the post extracted plasma samples from six different drugfree blank plasma samples, and tidy reconstitution samples, the matrix for Etoposide and Cisplatin with internal norm was evaluated. The examination was carried out in triplicate at MQC levels of six different plasma batches with the necessary accuracy (percent CV of) 15%.

Accuracy and precision
At lower concentration (LLQC), inferiority control (LQC), medium internal control (MQC), top quality control (HQC) stages, replicate analysis of internal control samples (n=6) was set. The %CV should be just 15 percent, and accuracy need not be within 15%, except that LLQC is within 20%.

Recovery
Study of six replicates with each internal control concentration determined the extraction ef iciencies of Etoposide and Cisplatin. The shared recovery has been estimated by separating the height ranges of the removed standards from those of the nonextracted standards.

Stability
Stability was maintained by differentiating the test area relationship and the internal norm within the stability sample, with the fresh stock solution being prepared for the sample. In the LQC and HQC absorption ranges, stability in plasma carried out using six times repeat at each step. The analyte was found to be stable if the shift is less than 15 percent according to the USFDA guidelines. The steadiness of the steady spike the temperature-stored samples (bench top stability) was analyzed for 24 hrs. The auto sampler stability (auto sampler stability) of spiked rat plasma tests collected at 2-8 • C was evaluated for 24 Hrs.  The stability of the auto sampler was calculated by differentiating the plasma extract samples injected immediately and re-injected into the samples after being held inside the auto sampler. Six lower and higher limit levels of the quality control sample collection quantity were obtained after 24 hrs for the calculation of absorption differentiating with the initial concentration.

Methods and Pharmacokinetics
Pharmacokinetic analysis was performed on six demonstrate the applicability of the proposed procedure. The institute of the animal ethics committee (Regd. No. 1074/PO/Re/S/07/CPCSEA) approved the animal protocol.

In the Vivo study design
Etoposide and cisplatin in six different rats were administered in oral dose under fasting condition. Rat plasma test samples were gathering into a tube through a cannula introduced into a tail vein of the rat with a volume of 0.2 ml to 0.4 ml at 15 min, 30 min, 45 min, 60 min, 90 min, 2 hrs, 24 hrs, 36 hrs, 48 hrs, 60 hrs, 72 hrs, 84 hrs and 96 hrs each sample was isolated and stored at 70 • C by centrifugation.

Optimization of conditions in chromatography
It has optimized the chromatographic conditions consistent with the experience of the techniques developed in hours for determining to realize the symmetrical form of the peak and an honest chromatographic solving for Etoposide and Cisplatin and therefore the blueprint internal standard within the shortest possible time. The utilization of the chromatographic system is connected. It was considered that a PDA detector was the estimation in Etoposide and Cisplatin in plasma samples for rats. Diverse conditions have been tested to seek out the simplest mobile phase, two different combinations of mobile

Method validation using HPLC and UPLC
A complete and detailed validation of etoposide and cisplatin it was done in rat plasma, in HPLC as well as UPLC as per USFDA guidelines. The method was checked in selective, sensitive, linearity, accuracy and precision, matrix condition, recovery study, reinjection stability and reproducibility.

Selectivity and prudence
Results to be obtained from scratch chromatogram and plasma peppered with a low level in quality control samples (LLQC). The mean deviation interference found during retention periods of etoposide and cisplatin between six separate plasma lots including K 2 EDTA contains lipidomic, and hemoly

Impact matrix
The percent RSD for inside the signal, ion suppression/enhancement was observed as 1.0 percent for etoposide and cisplatin in HPLC and 0.6 percent in UPLC at the MQC level, say that under these circumstances, the matrix impact on analyze ionization is within reasonable ionization range.

Precise and precise
The consistency and the precision were calculated by collecting anything all separate test outcome in reproducing the (n=6) internal over ive individual power group runs. The % CV in between run precision was < 5% and the value of inter run precision was in between 85-115 for etoposide and cisplatin. The results of precision and accuracy in HPLC and UPLC was tabulated in Tables 1 and 2 and their chromatograms are shown in Figures 3 and 4.

Alignment
The selected drug response calibration requirements ratios were corresponding to the focus of etoposide and cisplatin for each assay over the 5-100 ng/ml nominal concentration range of etoposide and 10-200 ng/ml of cisplatin for both HPLC Tables 3 and 4 and UPLC Tables 5 and 6. The curves of calibration seemed linear and were well-de ined by the method of minus squares rectilinear lines of regression Figures 5 and 6. The coef icient of correlation was ≥ 0.999 for Etoposide and Cisplatin.

Recovery
Six watery ones (sample spiked remedy for reconstitution) at a small, mean and top concentration of the quality management standards with etoposide and cisplatin was primed for determination on rehabilitation and therefore the areas accomplished for samples taken from an equivalent concentration from a precision the precision batch and the precision batch run on an equivalent day. Normal recovery for the etoposide and cisplatin in HPLC was 98.62% with a precision of 0.64%, and UPLC was 99.37% with the consistency of a 0.54% that means that the quality of extraction etoposide and cisplatin also oxalate consistent and was reproducible.

Re-injection reproducibility
Re-injection reproducible operation was carried out to examine instrument presentation stills constant following until the deactivation, regardless of instrument miscarriage through actual content typical investigation. The deviation was 2.1 at low, high levels of concentration; hence groups are often re-injected inside the case of device miscarriage through actual content typical investigation. Furthermore, the sample was prepared to be re-injected after 24 hrs just in case of malfunction of the instrument during a speci ic subject sample review.

Stability
The stock approach was carried out to see the security of Etoposide and Cisplatin available prepared options in diluents and stored in the fridge at 2-8 • C. In contrast, with stock solutions prepared before 24 hrs, freshly prepared stock solutions were compared the half of shift to Etoposide and Cisplatin was 1.21% and 0.89% in HPLC condition and 0.98%, 0.66% in UPLC which indicates stock solutions were safe, respectively minimum of for 24 hrs. Stability of bench top and auto sampler for Etoposide and Cisplatin both LQC and HQC levels were examined. Etoposide and cisplatin were in plasma safe for a minimum of 24 hrs at room temperature and 24 hrs at 20 • C in an auto sampler. It was corroborated that the plasma samples correlated with frequent freezing and thawing Etoposide and Cisplatin at LQC and HQC unaffected stability of theirs. Outcomes with long term peace indicate the one who etoposide and cisplatin were in a matrix stable up to 24 hrs at a factory temperature -30 • C. The stability results are shown in below Tables 7 and 8.

Pharmacokinetic studies
The supporting data has been fortunate to quantify etoposide and cisplatin concentrations in six groups of rats under abstain from food state after administration of the drug as an oral dose. After that, samples are prepared as per the test method and injected into both HPLC and UPLC chromatographic systems and record the values. The pharmacokinetic characteristics estimated were C max (highest seen drug accumulation during the steady), AUC 0−12 (peak response below the vital luid concentration, time loop measured, utilizing the rule of the trapezoid), tmax (time to Max seen the highest drogue accumulation), Kel (pictured terminal calculated constant of the irst-order rate from the half log graph of planetary plasma accumulation versus curve time, using the procedure regression of the least square) and t1 /2/2 (end half-way was calculated by using the blueprint formula 0.693 per Kel). The test reference relationships for C max , AUC 0−12 and AUC were 85.38%, 92.45% for HPLC and 88.16%, 95.38% for UPLC individually and these results were inside the allowable the 80-125 percent range shows bioequivalence and that formulation with this selected drugs. The Mean concentration vs time of selected drugs Etoposide and Cisplatin within rat plasma as test and reference Figures 7 and 8 the table is tabulated in Tables 9 and 10.

CONCLUSIONS
A quick, responsive and economic bio-analytical system for both HPLC and UPLC monitoring Etoposide and Cisplatin in the plasma of rat was evolved and located successfully and applicable for the bio availability of Etoposide and Cisplatin. The method developed had additional advantages relative to other methods of study of Etoposide and Cisplatin in the Samples from the plasma of rat. The indings of the validation studies also revealed that the optimized system of HPLC, as well as UPLC, has precision, sensitivity, and linearity, accuracy, and precision, stability, across the entire spectrum of extreme plasma therapeutic concentration.