Int J App Pharm, Vol 14, Issue 5, 2022, 53-61Original Article

PERAMIVIR AND RELATED IMPURITIES IN RAT PLASMA AND ITS APPLICATIONS IN PHARMACOKINETIC STUDIES (BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION BY LC-MS/MS)

THULASEEDHAR ALUMURI1, KARUNASREE MERUGU1*, NAMBURI L. A. AMARABABU2, ARAVIND KURNOOL3

1*Department of Chemistry, GITAM (Deemed to be University), Bengaluru 560034, Karnataka, India, 2New Generation Materials Lab (NGML), Department of Science and Humanities, Vignan’s Foundation for Science Technology and Research University (VFSTR) (Deemed to be University), Vadlamudi, Guntur 522213, Andhra Pradesh, India, 3Department of Chemistry, Osmania University, Hyderabad 500007, Telangana, India
*Email: kmerugu@gitam.edu

Received: 08 Jun 2022, Revised and Accepted: 05 Jul 2022

ABSTRACT

Objective: New LC-MS/MS method for the estimation of Peramivir and its associated substances was developed and validated

Methods: Optimized (Developed) method includes gradient elution of peramivir and its related substances with a flow of 1 ml/min and waters X-bridge C18 column of dimensions 150 mmx4.6 mm, 3.5µ. 0.1% formic acid and acetonitrile were used as the mobile phase. Sarilumab was used as an internal standard. 40 min run time was used to separate peramivir and its related substances.

Results: The calibration curve was linear in the concentration percentage range from 10%-200% of Peramivir and its related substances. The calibration charts plotted were linear with a regression coefficient of R2>0.999. Accuracy, precision, recovery, matrix effect and stability results were found to be within the suitable limits. A Simple and efficient method was developed and utilized in pharmacokinetic studies to see the investigated analyte in body fluids.

Conclusion: This application denotes all parameters such as accuracy, precision, recovery, stability etc, which are in good agreement with the USFDA guidelines and are effectively applied to the investigation of the pharmacokinetic studies in rat plasma.

Keywords: Peramivir, LC-MS/MS, Development, Validation, Rat plasma

© 2022 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2022v14i5.45457. Journal homepage: https://innovareacademics.in/journals/index.php/ijap

INTRODUCTION

Peramivir is a known analytical anti-viral drug [1, 2] usually treated in influenza [3, 4] related diseases, it has been developed by various Pharmaceutical companies. Peramivir is a neuraminidase inhibitor [5, 6] acting as a transition state analogue [7, 8] inhibitor of influenza neuraminidase, preventing viruses [9, 10] from emerging from infected cells. It has been approved for intravenous administration [11-13]. In the 2008-2009 intramuscular [14, 15] peramivir phase II seasonal influenza study, there was no effect for the primary end point of median improvement on the alleviation of symptoms in subjects with confirmed acute uncomplicated influenza infection versus placebo. On October 23rd, the US food and drug administration (FDA) [16] has issued an emergency use authorization for peramivir, allowing the use of an intravenous drug for hospitalized patients only in cases where other available treatment methods are ineffective or unavailable; for example when oseltamivir resistance develops and a person is unable to take zanamivir via the inhaled route [17]. The objective of this study is to develop and to validate the selected and sensitive LC-MS/MS method for the determination of peramivir in the plasma of rat and to gauge the pharmacokinetics of these compound after oral administration of exact samples in the rat. Fig. 1 shows the chemical structures of Peramivir and its related impurities.

Peramivir

Peramivir Imp-26

Peramivir Imp-12

 Peramivir Imp-20

Peramivir Imp-15

Peramivir Imp-7

Peramivir Imp-6

Peramivir Imp-1

Peramivir Imp-8

Peramivir Imp-5

Peramivir Trihydrate

Sarilumab

Fig. 1: Chemical structures of peramivir and its related impurities

In the present research, LC-MS was used for the simultaneous quantification of peramivir and its related impurities in rat plasma. Until now, there were no quantification methods for the estimation of peramivir and its related impurities. The present study was designed to investigate on (a) to create and approve a particular and delicate LC-MS/MS strategy regarding the assurance along with Peramivir and its related impurities plasma in rats, and (b) to assess the pharmacokinetics of these drugs after intravenous administration of test extracts in rats.

MATERIALS AND METHODS

Reagents (Chemicals and materials)

Reference standards for peramivir (99.9% purity) and its related impurities came from Cadila health care limited, Ahmedabad, India. HPLC marked acetonitrile; formic acid was obtained from Merck in Mumbai, India. HPLC grade Milli Q water is used for purification. (Milli Q system, USA).

Equipment

Waters alliance e-2695 model HPLC system was coupled to SCIEX QTRAP 5500 mass spectrometer with an electrospray ionization (ESI) interface [18, 19]. The SCIEX software [20-22] was used to interpret the chromatogram data. Column waters X-bridge C18 was used for separation and validation.

Conditions of the mass spectrometer

Multiple reaction monitoring (MRM) of the mass spectrometer with positive ion electrospray ionization mode (+ESI) was used for the separation of peramivir and its related substances. Collision energy of 15V and 14V, source temperature of 550 °C, ion spray voltage of 5500V, drying gas temperature of 120-250 °C, collision gas of nitrogen, inlet and outlet potential of 10V, 7V and Dwell time of 1 sec was used in mass spectrometer.

Conditions of chromatography

A mixture of 0.1% formic acid and acetonitrile was used as a mobile phase with gradient elution. 10 µl of injection volume and 1 ml/min of flow rate was used for this validation.

Standard solution preparation

By diluting with diluents, the standard solution of peramivir (50 ng/ml), imp-26 (10 ng/ml), imp-12 (5 ng/ml), imp-20 (10 ng/ml), imp-15 (10 ng/ml), imp-7 (10 ng/ml), imp-6 (5 ng/ml), imp-1 (20 ng/ml), imp-8 (15 ng/ml), peramivir trihydrate (active metabolite) (30 ng/ml), imp-5 (20 ng/ml), Sarilumab (internal standard) (50 ng/ml) were prepared. The standard solutions were stored at 4 °C and brought back to room temperature before use.

Table 1: Gradient program

Time (min) Acetonitrile Buffer (0.1% Formic acid)
0 20 80
10 50 50
20 70 30
30 20 80
40 20 80

Sample solution preparation

By adding 200 µl of plasma, 800 µl of acetonitrile, 500 µl of internal standard and 500 µl of standard stock, the sample solution was prepared. Mix in the vortex cyclomixture to precipitate all the proteins. Centrifuge for 20 min at 400 rpm, collect and inject the supernatant solution into the HPLC system.

Pharmacokinetic study

Selection of animals

In this study six healthy white albino rats (body weight between 250-350grams) were obtained from Biological E Limited, Hyderabad, India. The protocol of the animal study was approved by the institute of the animal ethics committee (Reg. No: 1074/PO/Re/S/05/CPCSEA). Six rats are under fasting condition. Blood samples were collected from cardiac puncture procedure. The rat is anesthetized and blood is collected via the left ventricle using a 19-21 gauge needle. Blood will be withdrawn slowly to prevent the heart from collapsing. vein with volume of 0.2 ml to 0.4 ml at 0, 0.3, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10 and 12 h. Each sample was separated by centrifugation and stored at-20 °C.

Method validation

The method was validated [23-31] in selective, sensitive, linearity, accuracy and precision, matrix condition, recovery study, re-injection reproducibility and stability.

Selectivity

The optimized LC-MS/MS method was determined by an analysis of 6 lots of individual rat plasma samples. Chromatograms of spiked rat plasma samples at the LLOQC level were compared with those of blank plasma samples.

Effect of the matrix

The matrix effect [32, 33] of rat plasma on the simultaneous analysis of peramivir was evaluated by comparing the peak area of peramivir in the extracted blank plasma with those of peramivir standard solution. It has been studied at three replicates of LQC and HQC levels.

Integrity of dilution

Integrity of dilution [34, 35] should be demonstrated by splitting the matrix with an analyte concentration above the ULOQC and diluting the sample with a blank matrix.

Accuracy and precision

Intraday precision and accuracy were tested in six replicates in a single set using samples of HQC, LQC, MQC and LLOQC concentrations. Inter-day precision and accuracy were tested by HQC, LQC, MQC and LLOQC in three separate batches. The accuracy was expressed as a percent CV and accuracy as percent recovery.

Carryover

Carryover [36, 37] is the small quantity of analyte present by the chromatographic system during the sample injection, which appears empty or unknown in subsequent samples.

Recovery

The extraction efficiency of peramivir was determined by an analysis of six replicates at each quality control concentration. The percentage recovery was assessed by comparing the peak areas of the extracted standards to the peak areas of the non-extracted standards.

Stability

Stability [38, 39] solutions were achieved by comparing the area response of the analyte in the stability sample with the area response of the sample prepared from the fresh stock solution. Plasma stability studies were conducted at HQC and LQC levels using six replicates at each level. The analyte was considered stable if the change is less than 15% as per USFDA guidelines. The stability of spiked rat plasma samples stored at room temperature (bench top stability) was evaluated for 24 h. The stability of the spiked rat plasma stored at 2-8 °C in the autosampler (autosampler stability) was evaluated for 24 h. The stability of the autosampler was evaluated by comparing the plasma extract samples that were immediately injected with the samples that were reinjected in the auto sampler for 24 h at 2-8 °C. Frozen thaw stability was achieved by comparing the stability samples frozen at-30 °C and thawed three fold with freshly spiked internal control samples. Six aliquots of each of the concentrations of LQC and HQC were used for the stability assessment of freeze-thaw. In the long-term stability assessment, the concentration obtained after 24 h was compared with the initial concentration.

RESULTS AND DISCUSSION

Electro sapray ionization (ESI) with maximum response over atmospheric pressure chemical ionization (APCI) mode selected by this method. Optimization of the instrument to provide sensitivity and signal stability during the continuous flow of the mobile phase analyte into the electrospray ion source operated at a flow rate of 10 µl/min at both polarities. Peramivir gives more response in positive ion mode when compared with negative ion mode.

Various columns such as C18, C8 and CN-propyl and mobile phases consisting of 0.1 percent formic acid and acetonitrile were tested to obtain the best chromatographic condition. The best chromatographic condition occurred in the waters symmetry C18 with a mobile phase of 0.1 percent formic acid and acetonitrile in gradient elution with a flow rate of 1 ml/min.

Fig. 2: Mass spectra of peramivir

Sensitivity

Blank plasma and spiked plasma with LOQ sample in of peramivir and its impurities. The percent interference of analyte retention time between six different batches of rat plasma, including hemolyzed and lipedemic plasma containing K2EDTA as an anti-peramivir coagulant, is within the acceptable criteria. Six replicates of extracted samples were prepared and analyzed at LLOQC level in one of the plasma sample with the least interference at peramivir retention time. The percent CV of the area ratios of these six replicates of samples was found to be within the acceptable limit.

Fig. 3: Blank plasma chromatogram of peramivir

Fig. 4: LLOQC chromatogram of peramivir and its related substances

Table 2: A and B are the linearity results of peramivir and its impurities

Table A

Linearity

Per conc

(ng/ml)

Per

res

 Imp26 Conc (ng/ml)

Imp26

res

 Imp12 conc (ng/ml)

Imp 12

res

 Imp20 conc (ng/ml) Imp 20 res Imp15 conc (ng/ml) Imp 15 res
Linearity-1 5 0.1 1 0.01 1 0.005 1 0.012 1 0.010
Linearity-2 12.5 0.25 2.5 0.025 2.5 0.013 2.5 0.030 2.5 0.025
Linearity-3 25 0.5 5 0.05 5 0.025 5 0.060 5 0.050
Linearity-4 37.5 0.75 7.5 0.075 7.5 0.038 7.5 0.090 7.5 0.075
Linearity-5 50 1 10 0.1 10 0.05 10 0.120 10 0.100
Linearity-6 62 1.25 12.5 0.125 12.5 0.063 12.5 0.150 12.5 0.125
Linearity-7 75 1.5 15 0.15 15 0.071 15 0.180 15 0.150
Linearity-8 100 1.957 20 0.19 20 0.096 20 0.236 20 0.189
Slope 0.02 0.00968 0.0048 0.0119 0.0096
Intercept 0.01 0.0015 0.0010 0.0006 0.0017
CC 0.99987 0.99926 0.99926 0.99992 0.99910

Table B

Linearity Imp7 conc (ng/ml)

Imp 7

res

Imp6

conc (ng/ml)

Imp 6

res

Imp1

conc (ng/ml)

Imp 1

res

Imp8

conc (ng/ml)

Imp

8 res

 Per hyd conc (ng/ml)

Per hyd

res

Imp5

conc (ng/ml)

 Imp 5 res
Linearity-1 1 0.012 0.5 0.002 2 0.035 1.5 0.020 3 0.050 2 0.035
Linearity-2 2.5 0.030 1.25 0.005 5 0.088 3.75 0.050 7.5 0.125 5 0.088
Linearity-3 5 0.060 2.50 0010 10 0.175 7.5 0.100 15 0.250 10 0.175
Linearity-4 7.5 0.090 3.75 0.015 15 0.263 11.25 0.150 22.5 0.375 15 0.265
Linearity-5 10 0.120 5 0.020 20 0.350 15 0.200 30 0.500 20 0.350
Linearity-6 12.5 0.150 6.25 0.025 25 0.438 18.75 0.250 37.5 0.625 25 0.438
Linearity-7 15 0.180 7.5 0.030 30 0.525 22.5 0.300 45 0.750 30 0.525
Linearity-8 20 0.232 10 0.038 40 0.661 30 0.377 60 0.948 40 0.662
Slope 0.0117 0.0039 0.0169 0.0128 0.0161 0.0169
Intercept 0.0012 0.0003 0.0061 0.0035 0.0078 0.0062
CC 0.99968 0.99926 0.99907 0.99902 0.99920 0.99909

Fig. 5: Linearity plot of peramivir

Fig. 6: Linearity plot of peramivir trihydrate

Fig. 7: Linearity plot of peramivir Imp-1, Imp-6, Imp-7 and Imp-8

Fig. 8: Linearity plot of peramivir Imp-5, Imp-26, Imp-12, Imp-15, Imp-20 and Imp-26

Matrix effect

The ion suppression/enhancement percentage of CV in the signal was found to be 0.1 percent in MQC levels of Peramivir. It indicates that the effect of the matrix [40, 41] on the ionization of the analyte is within the acceptable limit.

Linearity

It was clear from the calibration curve that the peak area ratios were proportional to the concentration. The peramivir and its related compound solutions were prepared in the concentration range of 10% to 200%. The calibration curve was linear and the correlation coefficient was found to be 0.999. The linearity results of peramivir and its related compounds were shown in the following table [42].

Precision and accuracy

By pooling all individual assay results of different internal control samples, the accuracy and precision [43] were calculated. It was obvious, based on the data provided, that the strategy was precise and effective. The precision results of Peramivir and its related substances were shown in table 3.

Recovery

For recovery determination low, medium and high-quality control concentrations for peramivir and its related substances have been prepared and the areas collected for extracted samples of the same concentration levels from a precision and accuracy batch run on the same day. The mean recovery of peramivir was 100.13 and the precision was 1.2 percent.

Carryover

System error, which may affect the measured value of the sample, is called carryover. Based on the following procedure was evaluated through LC-MS/MS system, which was configured by waters alliance. System blank injection of 10 µl, 0.1 percent formic acid and acetonitrile in gradient mode into the water Z spray triple quadrupole mass detector was performed using a flow injection analysis. From this, we can say that it does not affect the accuracy and precision of the method proposed. Sample carryover is expressed as percent carryover.

Reinjection and reproducibility

During the actual sample analysis, reinjection reproducibility was performed to check the device after hardware deactivation due to any instrument failure. At LQC and HQC levels, the change was less than 2.0 and therefore, the batch was reinjected in the case of instrument failure during the actual subject sample analysis. Samples were prepared and reinjected after 24 h showing that the percent change was less than 2.0 percent at LQC and HQC levels and; therefore, the batch can be reinjected after 24 h during the actual sample analysis in the event of instrument failure.

Stability

Peramivir and its related substances solutions were prepared and stored in a refrigerator at 2-8 °C for solution stability analysis. Fresh stock solutions were developed 24 h earlier in relation to aged stock solutions. It is clear that the sample solutions were stable up to 24 h by observing the values of peramivir and its related substances.

Peramivir was stable in plasma for 24 h at room temperature and in an autosampler at 20 °C for 24 h. It has been confirmed that repeating freezing and thawing of plasma samples spiked with peramivir and its related substances did not affect their stability at LQC and HQC. Long-term stability showed that peramivir was stable at a storage temperature of-30 °C for up to 24 h. In the following table, the overall stability results of peramivir were tabulated.

Table 3: Precision and accuracy results of peramivir and its related substances

Name Nominal conc (ng/ml) Within run Between run
Mean conc Standard deviation accuracy Mean conc Standard deviation Accuracy
Peramivir 5 4.98 0.214 99.8 4.99 0.207 99.6
25 25.01 0.748 100.1 25.02 0.726 100.2
50 49.99 0.362 98.9 50.01 0.384 99.9
75 75.02 0.159 100.2 74.98 0.147 98.9
Imp-26 1 0.99 0.854 98.7 1.01 0.868 99.7
5 4.98 0.462 99.6 5.02 0.496 100.1
10 10.02 0.153 99.9 9.98 0.151 98.6
15 15.01 0.524 100.1 14.99 0.572 98.8
Imp-12 0.5 0.51 0.274 99.8 0.49 0.213 99.9
2.5 2.52 0.163 100.1 2.51 0.108 100.1
5 4.99 0.584 98.7 4.98 0.574 99.6
7.5 7.51 0.721 99.9 6.99 0.698 98.7
Imp-20 1 0.99 0.639 98.6 1.02 0.619 100.2
5 5.01 0.310 99.9 5.02 0.313 100.1
10 9.98 0.527 98.8 9.99 0.557 99.8
15 15.02 0.495 100.2 14.98 0.478 98.9
Imp-15 1 1.01 0.837 100.1 0.99 0.816 99.7
5 4.98 0.754 98.5 5.01 0.743 100.1
10 10.02 0.778 100.2 10.01 0.778 99.9
15 14.99 0.637 99.7 15.03 0.613 100.2
Imp-7 1 0.98 0.485 99.4 0.99 0.441 99.5
5 5.02 0.129 99.9 4.98 0.126 99.9
10 9.99 0.384 98.9 10.01 0.396 100.1
15 15.01 0.754 100.1 15.03 0.778 100.2
Imp-6 0.5 0.51 0.298 99.9 0.49 0.283 99.7
2.5 l 2.52 0.854 100.1 2.48 0.821 98.8
5 4.99 0.085 98.8 5.01 0.159 99.9
7.5 7.48 0.074 98.5 7.51 0.084 100.1
Imp-1 2 1.96 0.845 99.4 1.98 0.831 99.6
10 10.01 0.374 99.9 9.99 0.352 99.8
20 20.02 0.473 100.1 20.02 0.496 100.1
30 29.98 0.985 99.6 29.97 0.867 99.6
Imp-8 1.5 1.49 0.821 98.8 1.51 0.881 99.9
7.5 7.51 0.364 100.1 7.48 0.352 98.7
15 14.96 0.874 99.3 15.01 0.745 99.9
22.5 22.53 0.855 100.3 22.49 0.766 98.5
Peramivir Trihydrate 3 2.98 0.827 99.6 3.01 0.859 100.1
15 15.01 0.638 99.9 14.99 0.662 98.9
30 29.99 0.096 98.7 30.01 0.145 100.1
45 45.02 0.381 100.1 44.98 0.372 99.8
Imp-5 2 2.01 0.874 99.9 1.97 0.866 98.7
10 9.99 0.772 98.9 10.02 0.735 100.2
20 19.94 0.193 98.3 19.97 0.167 98.9
30 30.1 0.589 100.1 29.98 0.553 99.8

mean±SD (n=6)

Fig. 6: Recovery plot of peramivir

Table 4: Stability results of peramivir and its impurities

Name Conc level Bench top stability Auto sampler stability Long term stability Freeze thaw stability Wet extract stability Dry extract stability Short term stability
mean±SD
Peramivir LQC 25.31±0.525 25.17±0.341 25.22±0.415 25.27±0.341 25.16±0.258 25.43±0.138 25.53±0.621
MQC 50.42±0.757 50.28±0.417 50.04±0.857 50.13±0.274 50.74±0.386 50.04±0.625 50.43±0.358
HQC 75.02±0.162 75.16±0.532 75.16±0.234 75.41±0.136 75.58±0.451 75.31±0.417 75.15±0.557
Imp 26 LQC 5.10±0.326 5.23±0.534 5.12±0.741 5.14±0.534 5.07±0.412 5.23±0.536 5.34±0.254
MQC 10.30±0.024 10.21±0.174 10.52±0.132 10.21±0.174 10.63±0.215 10.17±0.274 10.26±0.534
HQC 15.42±0.174 15.74±0.235 15.62±0.085 15.14±0.235 15.38±0.745 15.06±0.552 15.29±0.284
Imp 12 LQC 2.51±0.721 2.55±0.724 2.66±0.441 2.63±0.775 2.54±0.637 2.55±0.652 2.59±0.632
MQC 5.04±0.624 5.13±0.126 5.15±0.374 5.12±0.632 5.15±0.742 5.46±0.534 5.12±0.427
HQC 7.53±0.531 7.62±0.385 7.58±0.475 7.59±0.312 7.38±0.629 7.53±0.847 7.55±0.534
Imp 20 LQC 5.24±0.325 5.32±0.274 5.41±0.325 5.07±0.263 5.26±0.342 5.42±0.314 5.63±0.157
MQC 10.32±0.418 10.38±0.326 10.18±0.374 10.18±0.745 10.74±0.621 10.62±0.475 10.43±0.528
HQC 15.17±0.625 15.65±0.296 15.26±0.124 15.37±0.218 15.51±0.295 15.32±0.527 15.55±0.641
Imp 15 LQC 5.23±0.185 5.12±0.462 5.57±0.342 5.22±0.203 5.74±0.635 5.15±0.210 5.53±0.241
MQC 10.63±0.241 10.84±0.552 10.19±0.253 10.25±0.742 10.08±0.523 10.63±0.748 10.53±0.221
HQC 15.53±0.628 15.37±0.436 15.41±0.743 15.34±0.625 15.42±0.736 15.25±0.784 15.34±0.163
Imp 7 LQC 5.74±0.154 5.38±0.745 5.14±0.248 5.42±0.107 5.27±0.324 5.84±0.241 5.54±0.315
MQC 10.03±0.857 10.12±0.365 10.32±0.645 10.54±0.523 10.16±0.524 10.36±0.285 10.22±0.341
HQC 15.62±0.558 15.24±0.625 15.85±0.341 15.09±0.274 15.19±0.325 15.09±0.713 15.26±0.437
Imp 6 LQC 2.57±0.587 2.55±0.216 2.58±0.421 2.55±0.324 2.51±0.074 2.54±0.421 2.52±0.369
MQC 5.36±0.396 5.24±0.427 5.21±0.748 5.46±0.352 5.26±0.375 5.85±0.635 5.27±0.413
HQC 7.59±0.234 7.56±0.527 7.53±0.129 7.59±0.743 7.55±0.262 7.53±0.182 7.58±0.134
Imp 1 LQC 10.32±0.745 10.24±0.745 10.34±0.754 10.17±0.325 10.85±0.574 10.18±0.742 10.54±0.274
MQC 20.36±0.526 20.52±0.341 20.24±0.136 20.12±0.624 20.35±0.285 20.31±0.463 20.32±0.457
HQC 30.62±0.475 30.74±0.659 30.58±0.298 30.74±0.853 30.34±0.625 30.52±0.964 30.16±0.522
Imp 8 LQC 7.52±0.328 7.53±0.528 7.56±0.417 7.59±0.742 7.48±0.638 7.58±0.324 7.49±0.375
MQC 15.42±0.162 15.63±0.487 15.35±0.852 15.83±0.447 15.64±0.754 15.174±0.385 15.143±0.328
HQC 22.56±0.638 22.53±0.558 22.55±0.743 22.53±0.164 22.49±0.327 22.52±0.463 22.62±0.748
Peramivir Trihydrate LQC 15.32±0.745 15.27±0.321 15.64±0.425 15.64±0.354 15.47±0.328 15.23±0.524 15.65±0.632
MQC 30.56±0.857 30.36±0.421 30.35±0.124 30.18±0.689 30.24±0.748 30.57±0.882 30.14±0.174
HQC 45.23±0.857 45.54±0.856 45.18±0.746 45.74±0.362 45.06±0.819 45.34±0.642 45.36±0.842
Imp 5 LQC 10.24±0.748 10.15±0.749 10.32±0.685 10.74±0.698 10.34±0.457 10.39±0.547 10.63±0.742
MQC 20.56±0.235 20.45±0.843 20.64±0.487 20.35±0.241 20.63±0.285 20.48±0.352 20.08±0.421
HQC 30.41±0.624 30.55±0.624 30.35±0.648 30.49±0.374 30.17±0.241 30.49±0.748 30.39±0.052

mean±SD (n=6)

Table 5: Mean pharmacokinetic parameters of peramivir

Time (h) Mean response for 6-rats
0.0 0.00
0.3 0.578
0.5 0.954
0.75 0.784
1.0 0.610
1.5 0.480
2.0 0.350
4.0 0.287
6.0 0.140
8.0 0.070
10.0 0.000
12.0 0.000
Tmax 30 min
Cmax 0.954
T1/2 12H
AUC(0-t) 4 ng-h/ml
AUC(0-∞) 4 ng-h/ml
AUMC(0-t) 4 ng-h*h/ml
AUMC(t-∞) 315 ng-h*h/ml
AUMC(0-∞) 320-h*h/ml

Pharmacokinetic study

The liquid-liquid extraction method was used to isolate Peramivir in rat plasma. For this, 200 µl of plasma sample (respective concentration) were added into labelled polypropylene tubes and vortexed briefly; after that 300 µl of acetonitrile was added and vortexed for 10 min followed by centrifuged at 4000 rpm at 20 °C. After that, the separated aqueous layer was filtered with 0.45µ syringe filter.

Peramivir was administered as an oral dose under fasting condition of different groups of rats [44, 45]. After the drug samples are injected into the rat body [46, 47], the samples are collected at selected intervals of time, such as 30 min. After that, the samples were prepared as per the above procedure and injected into the chromatographic system and the values are recorded. The calculated accurate bioavailability of dosage of intravenous injection, Cmax after intravenous administration of Peramivir (0.954), Tmax (30 min), Kel (obvious first request terminal rate constant calculated from semi-log plot of plasma concentration versus time bend, using the least square relapse technique and t1/2 (terminal half-life as governed by 0.693/Kel quotient). Test/reference ratio for Cmax, AUC0-t and AUC0-∞ were 0.954, 4 ng-h/ml, 4 ng-h/ml, respectively, and found to be within the acceptable limit. Table 5 gives the Pharmacokinetic parameters [48, 49] of Peramivir.

CONCLUSION

The higher sensitive LC MS/MS method for the determination of peramivir in rat plasma has been developed and validated for the first time. In comparison to the protein precipitation method, we have developed liquid-liquid extraction for sample preparation with increased sensitivity as well as increased column life. The described method here is a robust, reproducible method of bioanalysis. Easy and systematic methods have been developed and can be used in pharmacokinetic studies and in the body fluids to check the analyte being examined.

ACKNOWLEDGMENT

I would like to thank my research supervisor for helping me in this study

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All authors have contributed equally.

CONFLICTS OF INTERESTS

The authors are conformed no conflicts of interest.

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