Development and Validation of two LCMS/MS Methods for Simultaneous Estimation of Oseltamivir and its Metabolite in Human Plasma and Application in Bioequivalence Study

 

Srinivasa Reddy, Nirmala Nayak, Imran Ahmed, Licto Thomas, Arindam Mukhopadhyay*, Saral Thangam

Norwich Clinical Services Pvt Ltd, 147/F, 8th Main, 3rd Block, Koramangala, Bangalore 560034, India

*Corresponding Author E-mail: arindam.mukhopadhyay@norwichclinical.com

ABSTRACT:

Oseltamivir phosphate is licensed for the treatment of patients with influenza virus infection. Two LCMS/MS methods for simultaneous quantification of Oseltamivir and its active metabolite, oseltamivir carboxylate in human plasma were described here. After solid phase extraction sample was separated either on a reversed phase C18 column with a stepwise gradient using 0.05% formic acid and methanol or a cation-exchange column using an isocratic mobile phase (7 mM Ammonium formate, pH 3.5 ± 0.2: Methanol: 50:50, v/v). Flow rate of 1 mL/min was maintained in both cases. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for detection and drug quantification. Both methods were validated over a range of 0.52ng/ml to 207.00 ng/ml for Oseltamivir and 4.08 ng/ml to 1200.00 ng/ml for Oseltamivir Carboxylate. Deuterated Oseltamivir and Oseltamivir carboxylate were used as internal standards. The accuracies and precisions for Oseltamivir were between 91-102% and 0.9 – 13.7% for all concentration levels. The accuracies and precisions for Oseltamivir carboxylate were between 88-109% and 0.5 – 8.2% at all levels. Furthermore, Oseltamivir and its metabolite were stable in plasma ex vivo for at least 191 days when stored at -20°C or below.

 

 

 

INTRODUCTION:

Influenza, a highly contagious, acute febrile respiratory viral infection, imposes a substantial disease burden on individuals at low and high risk for secondary illnesses. Differences in the core proteins have resulted in three serotypes of influenza virus – influenza A, B and C. Nearly all influenza-associated clinical illnesses are associated with Influenza virus A and B [1]. Recurrent epidemics with substantial human morbidity and mortality are caused by Influenza A viruses which are also associated with pandemics.

 

Influenza A has its pandemic origins in avian influenza viruses, the highly pathogenic H5N1 viruses. Principally a zoonosis, H5N1 is transmitted typically from fowl, or fowl product, to humans [2]. Recent evidence supports the occurrence of limited human to human transmission [3], while evidence from Pakistan supports human to human to human (third-generation) transmission [4]. Two glycoproteins, haemagglutinin and neuraminidase present on the surface of influenza virus, play important role for the virus life cycle. Haemagglutinin is responsible for the entry of the virus into cells of the respiratory epithelium, while sialidase activity in neuraminidase protein plays a critical role in the influenza virus replication cycle by facilitating the release of newly produced viral particles from infected cells. The design of neuraminidase protein inhibitors is currently one of the most common approaches in the development of anti-influenza virus drugs [5]. Oseltamivir phosphate, an ethyl ester pro-drug administered orally is the best known antiviral drug that slows the spread of influenza (flu) viruses (type A and B) between cells in the body.  It is rapidly and extensively metabolized by esterases in the gastrointestinal tract and liver to its active form, Oseltamivir carboxylate. It is a potent and selective inhibitor of the neuraminidase enzyme of the influenza viruses A and B. The sialidase activity of neuraminidase enzyme protein is responsible for cleaving sialic acid residues on newly formed virions and plays an essential role in the release and spread of progeny virions. When exposed to Oseltamivir, the influenza virions aggregate on the surface of the host cell, thereby limiting the extent of infection within the mucosal secretions and reducing viral infectivity. The drug is considered the best treatment for the bird flu disease (H1N1 and H5N1) [6,7].

 

Human plasma esterases can also cause variable and often extensive ex-vivo conversion of Oseltamivir Phosphate in blood and plasma samples under conditions likely to be encountered during clinical studies and during assay preparation. The esterase inhibitor dichlorvos prevents this conversion [8, 9]. Several methods have been reported for the determination of Oseltamivir and/or its active metabolite Oseltamivir carboxylate in different biological matrices [10 –19]. However, these methods suffer from several drawbacks like lack of assessment of matrix effect [10, 17, 19], no extended batch verification [10–17,19] or not determination of stock solution stability [10 –13, 17]. Owing to these problems a regulatory compliant method becomes essential for Oseltamivir estimation. Two methods have recently been described [15, 20]. These methods have been validated as per US FDA requirements. However, these methods have used the esterase inhibitor, dichlorvos, to prevent the conversion of Oseltamivir phosphate to Oseltamivir carboxylate. But the use of organophosphate compounds in the laboratory raises safety issues [9]. Keeping this in mind we developed new method for estimation of Oseltamivir and its metabolite using two different LCMS/MS conditions. Instead of health hazardous esterase inhibitor dichlorvos, a sodium fluoride – potassium oxalate vacutainer tube has been routinely used to collect the blood sample. These methods have been validated as per US FDA requirements [21]. One of these methods has also been tried in a bio-equivalence study for quantitation of Oseltamivir and its metabolite.

 

MATERIALS AND METHODS:

Chemical and Reagents:

Oseltamivir phosphate (purity: 99.5%) and its metabolite, Oseltamivir carboxylate (purity: 98.16%) were purchased from USP/LGC Promochem and MSN Pharmachem Pvt. Ltd, respectively. Oseltamivir D5 HCl (purity: 98.72%) and Oseltamivir carboxylate 13C D3 (purity: 99.12%) used as internal standards were purchased from Clearsynth and Vivan Life Sciences, respectively. Methanol (HPLC-grade), ammonium formate, ortho phosphoric acid and formic acid were of analytical grade and purchased locally. Milli Q purified water (Millipore, Milford, MA) was used throughout the study.  Blood was collected from adult, healthy volunteers in a vacutainer containing sodium fluoride and potassium oxalate. Plasma was separated by centrifugation at 3000 g for 10 min, and kept frozen at -70^oC until analysis.

 

Sample Preparation:

After vortexing, 100 μl of thawed sample was transferred to a new RIA vial. 50 μl of mixture of internal standards (Oseltamivir D5+Oseltamivir Acid 13C D3) was then added and mixed. 500 µl of 10% Ortho Phosphoric acid was added to the vial and vortexed. After centrifugation at 4000 rpm for 5 min at 4°C the sample was loaded onto a pre-conditioned solid phase cartridge (Phenomenex strata-33µ polymeric reversed phase, 30mg/1ml) kept in a RIA vial. The cartridge was pre-conditioned with 1ml Methanol followed by 1 ml of Milli Q water. RIA vial with loaded cartridge was centrifuged at 1000 rpm for 5 min at about 4°C and discarded the eluent. 1ml of 10% Ortho Phosphoric acid was added to the cartridge and centrifuged at 2000 rpm for 5 mins at about 4°C. The eluent was discarded. Similarly the cartridge was washed with 1 ml of 5% Methanol twice and discarded the eluent. The sample was then eluted by adding 1 ml of Elution solvent (0.1% Formic Acid: Methanol:: 20:80, v/v) to the cartridge and centrifuging at 500 rpm for 5 mins at about 4°C. The eluent was collected in a fresh RIA vial. It was then transferred to the labeled HPLC vial and loaded into auto sampler and injected. The whole process of sample preparation was performed in ice cold condition.

 

Chromatography and Mass Spectrometry:

Method A: Reverse Phase Chromatography:

20 µl of sample was injected to Zorbax SB-C8, 4.6×100 mm, 3.5 µm (Agilent Technologies) for separation. The column oven temperature was maintained at 50°C. Run time was 6.0 min. Following gradient method was employed for sample separation:

 

 

Time(min)	Flow rate (ml/min)	0.05% Formic Acid (% A)	Methanol (% B)
Initial	1.000	65	35
3.50	1.000	65	35
3.51	1.300	45	55
5.00	1.300	45	55
5.01	1.000	65	35
6.00	1.000	65	35

 

 

Mass spectrophotometric detection was performed on LCMS/MS API 4000 (AB Scientific Applied Biosystems) in positive mass ionization mode with multiple reaction monitoring (MRM) using an electrospray interface (ESI). Oseltamivir and Oseltamivir D5 were detected using selective reaction monitoring of the specific transitions m/Z 313.3>166.1 and 318.3>171.2, respectively. Similarly, Oseltamivir carboxylate and Oseltamivir carboxylate (13C D3) were detected following m/z transitions from 285.3>138.1 and 289.2>138.1, respectively. The collision energy and cone voltage were optimized for each analyte to maximize the signal corresponding to the major transition observed in the MS/MS spectra, following the fragmentation of the [M+H]⁺ ions corresponding to the selected compounds. The ion source parameters were the following: CUR 30 psi; GS1 45 psi; GS2 55 psi; CAD 8 psi; ion spray voltage (ISV) 5000 V; dwell time 500msec and temperature 400⁰C. The other parameters for both Oseltamivir and Oseltamivir carboxylate were: declustering potential (DP):  50 V, entrance potential (EP): 10 V, collision energy (CE): 25 V and cell exit potential (CXP): 10 V.

 

Method B: Ion – exchange Chromatography:

20 µl of sample obtained from SPE method was injected to a Bio-basic SCX column (50x4.6mm, 5µm) (Thermo Scientific) and was eluted with an isocratic mobile phase (7 mM Ammonium formate, pH 3.5±0.2: Methanol: 50:50, v/v)at a flow rate of at 1 mL/min. Total run time was 4.5 min. Column oven temperature was 40°C. The ion source parameters were as follows: CUR 30 psi; GS1 45 psi; GS2 55 psi; CAD 8 psi; ion spray voltage (ISV) 5000 V; temperature 400⁰C and dwell time 500msec. The other parameters for both Oseltamivir and Oseltamivir carboxylate were: declustering potential (DP):  60 V, entrance potential (EP): 10 V, collision energy (CE): 25 V and cell exit potential (CXP): 13 V. Oseltamivir and its metabolite were detected along with the respective internal standards using selective reaction monitoring of the specific transitions of m/z as mentioned earlier.

 

Calibration Standards and Quality Control (QC) Samples:

a)    For Reversed Phase (RP) chromatography: Oseltamivir stock solution concentration in methanol (200µg/ml) was further corrected by accounting for its potency and actual amount weighed to obtain the final concentration. Stock solution of Oseltamivir was serially diluted to prepare working solutions in the required concentration range with diluent (methanol–water 50:50, v/v). The calibration standards were prepared by spiking plasma with working solutions yielding concentration range from 0.517ng/ml to 206.862ng/ml for Oseltamivir and 4.079ng/ml to 1202.421ng/ml for Oseltamivir carboxylate.

 

Oseltamivir QC stock solutions have been prepared separately in methanol– water (50:50, v/v). QC working samples for Oseltamivir at four different levels: lower limit of quantitation (LLOQ)- 0.523ng/ml, low quality control (LQC) (LQC, within three times of the LLOQ)- 1.376ng/ml, middle quality control (MQC)- 66.141ng/ml and high quality control (HQC) - 157.479ng/ml, were prepared from this stock solution by dilution.  Similarly, calibration standards for Oseltamivir carboxylate were prepared in the concentration range of 4.079ng/ml to 1202.421ng/ml. Four QC samples for Oseltamivir carboxylate, LLOQ- 4.130ng/ml, LQC-11.162ng/ml, MQC-372.077ng/ml, HQC-930.192ng/ml were also prepared.

 

b)   For Ion-exchange chromatography:

The calibration standards were prepared in a similar way in the concentration range from 0.520 to 206.566 ng/ml for Oseltamivir and 4.080 to 1199.535 ng/ml for Oseltamivir carboxylate.  QC samples at four different levels: lower limit of quantitation (LLOQ)- 0.532 ng/ml, low quality control (LQC, within three times of the LLOQ) - 1.401 ng/ml, middle quality control (MQC) - 66.727 ng/ml and high quality control (HQC) - 158.873 ng/ml, for Oseltamivir and LLOQ - 4.137 ng/ml, LQC - 11.181 ng/ml, MQC - 372.694 ng/ml , HQC - 931.735 ng/ml for Oseltamivir carboxylate were prepared.  The stock solution of internal standards containing Oseltamivir D5 (200µg/ml) and Oseltamivir carboxylate 13C D3 (200µg/ml)was diluted using methanol: water (50:50) to a final solution containing 100ng/ml for Oseltamivir D5 and 2µg/ml for Oseltamivir Acid 13C D3.

 

Validation of the Bioanalytical Method

Both the methods were validated as per US-FDA guideline in human plasma [21]. The Reversed Phase (RP) method was evaluated for linearity of response, specificity and selectivity, sensitivity, precision and accuracy, recovery, stability of analytes and dilution integrity during both long-term storage and short-term sample processing for RP processing whereas for ion-exchange chromatography linearity of response, specificity and selectivity, precision and accuracy, long-term stability of sample have been evaluated. Matrix effect and carry over test were performed for both chromatographic conditions.

 

Bioequivalence study

A bioequivalence study has been conducted for a test product (Oseltamivir tablet) and a reference marketed product as per the pre-approved protocol and other documents by the Independent Ethics Committee (I.E.C. Consultants, Bangalore, India; EC Regn. No. ECR/60/Indt /KA/2013; validity: 3 years). 72 healthy human subjects had been recruited for this study based on the inclusion and exclusion criteria mentioned in the protocol. Informed consent procedure was conducted as per the regulations. Good Clinical Practice (GCP) and the applicable principles of Good Laboratory Practices (GLPs) and other Rules and Regulations were strictly followed for conducting this study.

 

RESULT AND DISCUSSION:

Sample preparation step before LCMS/MS plays an important role in producing clean samples. This helps to prevent the clogging of the analytical column and also to minimize ion –suppression/enhancement and matrix effect in the later step. Liquid –liquid extraction (LLE) or solid phase extraction technique (SPE) can produce the clean samples devoid of any endogenous substance to interfere with the analysis and functioning of LC and MS/MS systems. However, these extraction techniques are time consuming, costlier and also use more organic solvents than the simpler and cost effective precipitation method; but, SPE is superior to others in terms of producing cleaner sample with relatively less matrix effect and compared to LLE uses less solvent. We used Phenomenex strata-33µ polymeric reversed phase (30mg/1ml) cartridge during SPE process.

 

Matrix Effect:

The matrix effect (co-eluting, undetected endogenous matrix compounds that may influence the analyte ionization) was evaluated for eight lots of blank matrix including one heamolysed and one lipemic lot from different sources using either a reversed phase or an ion-exchange column. 100 μL of blank plasma from each lot was processed as mentioned earlier under the ‘Sample Preparation’. Each processed sample was then spiked with analyte mixture containing Oseltamivir and Oseltamivir carboxylate either at LQC or HQC level and also with intended concentration of IS. These samples were considered as post – extracted samples. Similarly, the aqueous solutions of Oseltamivir and its metabolite at LQC or HQC level along with intended concentrations of internal standards added to elution solvent were considered as aqueous samples. Six replicates of each aqueous sample were injected along with post extracted samples of LQC and HQC either to a RP or a SCX column.

 

Individual analyte area response and corresponding IS area response of each post extracted sample were compared with the mean analyte area response and mean respective IS area response of the aqueous sample respectively. The matrix effect was calculated via the formula: Matrix effect (%) = A₂/ A₁ x 100 (%), Where A₁= response of aqueous concentrations and A₂ is response of post-extracted concentrations. From the calculations, it was observed that in case of SCX column Oseltamivir showed an average (n=6) matrix factor of 115.936% at LQC level with a CV of 2.97% and 110.358%at HQC level with a CV of 1.24% which are within the accepted limit (% CV ≤15) (Table- 1). For Oseltamivir carboxylate, average matrix factor at LQC level was 100.815% with a CV of 6.27% and 100.034% at HQC level with a CV of 1.03%. A comparable value was obtained for the RP column also (Table – 1). This clearly indicates that using Phenomenex strata-33µ polymeric reversed phase (30mg/1ml) cartridge during SPE process resulted in clean samples which did not show any matrix effect irrespective of columns used during LCMS.

 

 

Table – 1: Matrix Effect for Oseltamivir and Oseltamivir Carboxylate

 

Table 2: Specificity and Selectivity of Oseltamivir and its metabolite

 

Specificity and Selectivity:

Human blank plasma lots were assessed for the selectivity of the method and plasma lots showing negligible or no interference at the retention time of internal standards and analytes. Ten different lots of blank matrix including one haemolysed and one lipemic lot (used for specificity) were spiked with LLOQ and intended concentration of internal standards (100ng/ml for Oseltamivir D5 and 2µg/ml for Oseltamivir Acid 13C D3) and processed. Interferences at the retention times of analytes and IS were evaluated by comparing peak area response with that of blank plasma. Signal to noise ratio for all lots was more than 5.0 indicating the method is selective for both Oseltamivir and its metabolite (Table - 2).For specificity, the interference should be ≤20% of LLOQ area response of analyte and ≤5% of the IS area response when compared to the selectivity area response of the respective lots. There was no interference observed in the screened lots of biological matrix for both Oseltamivir and Oseltamivir carboxylate. Therefore the methods described here were both specific and selective for Oseltamivir and its metabolite, Oseltamivir carboxylate.

 

Carry Over Effect

Carryover effect was evaluated to ensure that the rinsing solution used to clean the injection needle and port was able to avoid any carry forward of injected sample in subsequent runs. The design of the experiment comprised LLOQ, blank plasma, upper limit of quantization (ULOQ) followed by blank plasma to check for any possible interference due to carryover. There was no carry over observed during the study.

 

Chromatography:

As per FDA guidelines, internal standards should be preferably identical to the analyte (19) and hence labeled internal standards were used as the reference compounds in both the chromatographic conditions as mentioned below:

 

a)   Reverse Phase Chromatography:

Retention times for both Oseltamivir and Oseltamivir D5 were4.85 and 4.82 (Fig.1 a and b). Similarly, retention times for Oseltamivir carboxylate and its IS were2.59 and 2.56(Fig. 2 a and b). No interfering peaks were observed in the blank at the retention times corresponding to both analytes and respective IS indicating that the procedure is specific to Oseltamivir and its metabolite.

 

b)   Ion – exchange Chromatography:

In SCX column, Oseltamivir and Oseltamivir D5 had retention times 3.76 and 3.73, respectively (Fig.3 a and b). Similarly, retention times for Oseltamivir carboxylate and its IS were 2.38 and 2.36 (Fig. 4aand b). The method was specific for Oseltamivir and its metabolite as there was no interfering peaks in the blank at the retention times corresponding to both analytes and IS.

 

 

 

 

Fig. 1(a)                                                                                                                            Fig.1(b)

Figure 1 a and b: Chromatograms of Oseltamivir (1a) and IS (1b) in C18 Column chromatography

 

 

Fig. 2(a)                                                                                                        Fig. 2(b)

Figure 2 a and b: Chromatograms of Oseltamivir carboxylate (2a) and IS (2b) in C18 Column chromatography

 

 

Fig. 3(a)                                                                                                             Fig. 3(b)

Figure 3 a and b: Chromatograms of Oseltamivir (3a) and IS (3b) in SCX column chromatography

 

 

Fig. 4(a)                                                                                                             Fig. 4(b)

Figure 4 a and b: Chromatograms of Oseltamivir (4a) and IS (4b) in SCX column chromatography

 

Limit of detection

To find out the detection limit stock solution was diluted based on LLOQ area response to a concentration 0.259ng/ml (A/2) and 0.129ng/ml (A/4) for Oseltamivir. Similarly, for Oseltamivir carboxylate dilutions to 2.040ng/ml (A/2) and 1.020ng/ml (A/4) were made. The above dilution was spiked into matrix in triplicates, and processed along with LLOQ sample in triplicates.

 

Limit of detection was determined only for method using RP column and for Oseltamivir, it was 0.129ng/ml with signal to noise ratio more than 39. Similarly, limit of detection for Oseltamivir carboxyl ate was 1.020ng/ml with signal to noise ratio more than 83.

 

Sensitivity:

To determine sensitivity of the method six samples of LLOQ (STD A) were processed and were injected along with the PA. This method for simultaneous quantification of Oseltamivir and Oseltamivir carboxylate was found to be very sensitive even for very low concentration (LLOQ) of 0.517ng/ml with precision of 2.85% and accuracy of 109.32% for Oseltamivir and 4.079ng/ml with precision of 3.42% and accuracy of 109.86% for Oseltamivir carboxylate.

 

Precision and Accuracy:

Precision and accuracy of this method was determined with six replicates of quality control samples at different concentrations containing known concentrations of analyte within the concentration range of bio-analytical method. One set of calibration curve samples was also included. At least 3 precision and accuracy batches were run on to prove that the test method is precise and accurate. One PA batch (PA-4) was processed and run in different system as ruggedness PA. Precision and accuracy were calculated in terms coefficient of variation (%CV). At each concentration level a deviation within ± 15.0% from the nominal concentration was acceptable except LLOQ, for which it should be within ± 20.0%. Minimum 67% (4 out of 6) of the quality control samples at each level should meet the acceptance criteria. Table – 3and 4 (a and b) showed the mean accuracy and precision for intra- (within run) and inter-day (between) batches.

 

 

 

Table – 3a: Precision and Accuracy of method for calibration standards of Oseltamivir

Sample ID	Reversed Phase (C18) Column					Cation exchange (SCX) Column
	Nominal conc. (ng/ml)	Mean conc. (ng/ml)	SD	Mean accuracy (%)	%CV	Nominal conc. (ng/ml)	Mean conc. (ng/ml)	SD	Mean accuracy (%)	%CV
STD A	0.52	0.51	0.01	98.61	2.01	0.52	0.52	0.02	99.68	3.61
STD B	1.03	1.06	0.04	102.76	3.85	1.04	1.05	0.07	100.67	7.04
STD C	12.31	12.25	0.16	99.50	1.30	11.06	11.01	0.12	99.46	1.06
STD D	30.78	31.11	0.38	101.06	1.22	30.74	31.03	0.12	100.97	0.39
STD E	61.56	62.34	0.92	101.26	1.48	61.47	61.86	0.94	100.63	1.52
STD F	102.60	101.96	1.21	99.37	1.19	102.46	102.01	1.38	99.56	1.35
STD G	165.49	162.94	3.94	98.46	2.42	165.25	164.86	2.55	99.76	1.55
STD H	206.86	204.74	3.40	98.98	1.66	206.57	205.05	1.19	99.27	0.58

 

 

 

Table – 3b: Precision and Accuracy of method for quality control samples of Oseltamivir

Parameters	Reversed Phase (C18) Column				Cation exchange (SCX) Column
	LOQQC	LQC	MQC	HQC	LOQQC	LQC	MQC	HQC
Within run mean accuracy (%)	111.28	97.77	96.24	95.58	100.03	99.04	97.33	97.13
Within run mean precision (%)	6.45	5.82	2.35	1.23	13.07	3.89	1.09	1.40
Between batch mean  accuracy (%)	103.24	98.88	98.38	98.56	96.04	98.64	97.72	99.37
Between batch mean precision (%)	6.51	4.33	2.41	2.19	11.99	4.54	2.52	2.83

 

 

 

Table - 4a: Precision and Accuracy of method for calibration standards of Oseltamivir carboxylate

Sample ID	Reversed Phase (C18) Column					Cation exchange (SCX) Column
	Nominal conc. (ng/ml)	Mean conc. (ng/ml)	SD	Mean accuracy (%)	%CV	Nominal conc. (ng/ml)	Mean conc. (ng/ml)	SD	Mean accuracy (%)	%CV
STD A	4.08	4.10	0.05	100.42	1.12	4.08	4.15	0.07	101.61	1.71
STD B	8.16	8.09	0.18	99.14	2.27	8.16	7.75	0.21	94.99	2.68
STD C	71.57	71.39	1.39	99.75	1.95	64.26	64.47	0.49	100.34	0.76
STD D	178.92	179.91	2.10	100.55	1.17	178.49	180.76	1.49	101.27	0.83
STD E	357.84	363.13	6.96	101.48	1.92	356.98	361.58	2.09	101.29	0.58
STD F	596.40	596.71	13.30	100.05	2.23	594.97	593.16	8.50	99.70	1.43
STD G	961.94	954.59	12.16	99.24	1.27	959.63	965.92	21.76	100.66	2.25
STD H	1202.42	1194.81	15.14	99.37	1.27	1199.53	1181.30	9.92	98.48	0.84

 

 

 

Table – 4b: Precision and Accuracy of method for quality control samples of Oseltamivir carboxylate

Parameters	Reversed Phase (C18) Column				Cation exchange (SCX) Column
	LOQQC	LQC	MQC	HQC	LOQQC	LQC	MQC	HQC
Within run mean accuracy (%)	93.91	94.65	95.59	95.04	88.09	97.12	98.84	96.85
Within run mean precision (%)	3.90	6.05	2.37	0.98	8.21	2.30	0.54	1.45
Between batch mean accuracy (%)	95.06	95.91	96.37	95.54	99.07	98.63	96.03	97.78
Between batch mean precision (%)	4.35	3.15	1.93	1.32	10.75	4.74	3.41	2.56

 

 

 

 

As per FDA guidelines the within run and between run percent accuracy should be within ±15% of the actual concentration for each quality control sample except for LOQQC.  For LOQQC, it should be within ±20%.The within run and between run precision (%CV) should be less than or equal to 15% for each QC except for LOQQC for which it should be less than or equal to 20%.

 

Thus the result given in Tables–3 (a and b) and 4 (a and b) showed that both the methods were precise and accurate.

Linearity of the Calibration Plot

Calibration curves indicated a linear graph for Oseltamivir in the concentration ranges of 0.517ng/ml to 206.862ng/ml (Fig. 5a) and for Oseltamivir carboxylate in the range of 4.079ng/ml to 1202.421ng/ml (Fig. 5b). Residual sum of squares were obtained to check the large linear ranges used for this drug. The concentration of unknown was calculated from the equation y= mx +c using regression analysis of spiked plasma calibration standards with reciprocal of the square of the drug concentration (1/X²).

 

 

 

Fig. – 5(a): Calibration curve for Oseltamivir

 

 

 

 

Fig. – 5(b): Calibration curve for Oseltamivir carboxylate

 

Recovery:

Absolute recovery percentage was determined by comparing the mean peak area of analyte obtained by injecting 6 extracted samples of LQC, MQC and HQC with the mean peak area obtained by injection of respective aqueous standard solutions. In this method overall recovery for Oseltamivir was 89.5% with a %CV of 11.14 which is well within 15% (Table 5). Recovery for Oseltamivir carboxylate was 85% with a CV of 1.28%.

 

Table – 5: Recovery of Oseltamivir and Oseltamivir carboxylate from Biological Matrix

Oseltamivir
	LQC	MQC	HQC	Average	%CV
% Recovery	77.98	95.33	95.17	89.49	11.14
Oseltamivir carboxylate
% Recovery	83.80	85.98	84.95	84.91	1.28

 

 

Stability studies:

Short – Term/bench -top stability (STSS):

To determine the short term stability, stock solutions of analyte was prepared separately and kept at room temperature.MQC concentrations of both analytes were then prepared by stock dilution and stored at 25⁰C for 32 hrs. Six replicate injections were given for MQC samples. No significant differences were noticed when these results were compared with those obtained from the freshly prepared MQC samples indicating that Oseltamivir and its metabolite, Oseltamivir carboxylate were stable at room temperature (Table 6). Accepted criteria for the ratio of mean response for stability samples should be between 90-110%.

 

Long term stock solution stability (LTSS)

LTSS is performed to ensure that the analyte is stable at 2-8⁰C. Aqueous MQC sample was prepared from stability stock stored at 2-8 ⁰C for 10 days and injected. Mean area response of MQC of stored stock solution was then compared against MQC from freshly prepared stock solution. Mean percent stability for Oseltamivir and Oseltamivir carboxylate were 100.01% and 105.14% respectively which were well within accepted limit (90 – 110%) indicating that solutions of Oseltamivir and its metabolite were stable for 54 days at 2-8 ⁰C (Table 6).

 

 

 

 

Table – 6: Stability Studies of Aqueous Stock Solutions

Stability Check	Samples	Average Area for stored Solution	Average Area for Fresh Solution	% Stability
Oseltamivir
STSS  (32hrs)	MQC (n=6)	262968.7	258115.8	102.29
LTSS  (54 days)	MQC (n=6)	235776.7	238499.7	100.01
Oseltamivir  carboxylate
STSS  (32hrs)	MQC (n=6)	330389.2	321812.8	100.35
LTSS  (54 days)	MQC (n=6)	304385.7	322228.3	105.14

 

 

 

 

 

Stability in biological matrix

Bench-top stability:

To check whether the sample is stable during analysis, six aliquots of LQC and HQC samples were thawed and kept at room temperature (25⁰C) for 8hrs, which has been decided based on the time required for analysis. The samples were then processed and analyzed as mentioned above. The % stability was 98.11 for LQC and 98.54 for HQC (well within the accepted limit of 85-115%) indicating that Oseltamivir was stable at room temperature (Table– 7a). Similarly for Oseltamivir carboxylate, stability was 103.11% for LQC and 99.2% for HQC (Table – 7b).

 

Auto-sampler stability:

The stability of the processed samples in the auto sampler during analysis was determined by using six aliquots of LQC and HQC samples. The stability of Oseltamivir was assessed for 23 hours and 50 mins, the expected run time for batches of validation samples. The result was then compared with those obtained from fresh QC samples. For IS in-injector stability, the IS/analyte area ratio of MQC stored in auto sampler for same time was compared against freshly prepared MQC samples. No significant difference in the results indicated that the analytes and IS were stable for at least 23 hours and 50 mins in the auto sampler (Table– 7a). Similar experiment with Oseltamivir carboxylate and respective IS also indicated their stability (Table – 7b).

Freeze thaw stability:

Stability of analytes was determined after four freeze thaw cycles for six aliquots of each of the LQC and HQC. The samples were stored below – 70⁰C for 24h and then allowed to thaw at room temperature. After complete thawing, the samples were again stored at same temperature (– 70⁰C) for 12h. The freeze thaw cycle was repeated another three times before analyzing the samples. After 4 freeze thaw cycles, the above samples were processed and analyzed along with eight freshly spiked calibration standards and six aliquots each of comparison samples of HQC and LQC. No differences were noticed when the results were compared with the fresh QC samples indicating the stabilities of Oseltamivir and its metabolite in human plasma for four freeze thaw cycles (Table –7a and 7b)

 

Wet Extract stability:

To check whether the sample is stable after processing, six aliquots of LQC and HQC samples were processed and kept at room temperature for 5 hours. The samples were then analyzed as mentioned above. No significant differences were noticed when these results were compared with those obtained from the fresh QC samples indicating that processed samples of Oseltamivir and Oseltamivir carboxylate were stable at room temperature (Table 7a and 7b)

 

 

 

 

 

Table – 7a: Stability Studies of Oseltamivir

 

 

 

 

Table – 7b: Stability Studies of Oseltamivir Carboxylate

 

 

Long term stability

Long term stability is performed to ensure that the analyte and its metabolite are stable in matrix under the predefined storage conditions.

 

6 aliquots each of LQC and HQC samples bulk spiked along with other QC samples were stored either at about -70^oC or below -20^oC. After 191 days these samples were removed from the freezer and allowed to thaw. These samples were analyzed by LCMS/MS using SCX column against eight freshly spiked calibration standards and six aliquots of freshly spiked samples of LQC and HQC. The percentage mean concentration for stability samples should be within 85% to 115% compared to mean concentration of freshly prepared samples.

 

 

 

Table – 8:Long term stability of Oseltamivir and its carboxylate for 191 days

Analytes	Storage Temp 0C)	LQC				HQC
		Nominal conc. (ng/ml)	Mean conc. obtained (ng/ml)	% Stability	% CV	Nominal conc. (ng/ml)	Mean conc. obtained (ng/ml)	% Stability	% CV
Oseltamivir	- 200C	1.38	1.29	93.55	7.92	157.48	152.38	96.37	2.20
Oseltamivir Carboxylate		11.16	10.68	89.68	5.42	930.19	854.88	92.82	1.99
Oseltamivir	- 700C	1.38	1.44	104.24	3.71	157.48	162.54	102.80	1.50
Oseltamivir Carboxylate		11.16	11.41	95.79	4.55	930.19	892.73	96.93	0.89

 

The result given above indicated that both Oseltamivir and its metabolite, Oseltamivir carboxylate were stable for 191 days when stored either at -20⁰C or -70⁰C.

 

Dilution Integrity:

In order to validate the dilution test, dilution integrity experiment was carried out on higher analyte concentrations (above ULOQ), which may be encountered during real subject samples analysis. Stock dilution at twice the concentration of HQC was spiked into plasma and aliquots were prepared with the same and stored at about -70°C until analysis.

 

Dilution integrity samples were prepared by diluting 2 fold and 4 fold with interference free biological matrix. Six replicates of these samples were processed and analyzed against a set of freshly spiked calibration standards. If  % nominal was within ± 15 % (±20% in case of LLOQ) of nominal values and % CVs ± 15 % at both diluted levels, the integrity of the samples were considered to be maintained.

 

For 2 fold (2T) and 4 fold (4T) dilutions of Oseltamivir, accuracy were 103.93% and103.90% whereas precision were 1.03%  % and 1.62% respectively (data not shown). Similarly, for 2T and 4T dilutions of Oseltamivir carboxylate accuracy were 104.47% and 106.47% whereas precision were 1.04% and 0.81%.

 

Bioequivalence study:

The validated method using SCX column was applied to a bioequivalence study to compare a ‘test’ product to that of ‘marketed’ product among healthy volunteers in India. The study was conducted as per the regulations and prior approval for the study was obtained from an independent ethics committee. Oseltamivir and its metabolite in human plasma were simultaneously quantitated by using this method. The pharmacokinetic parameters were then calculated from data generated by this LCMS/MS method (data not shown). The ratio and 90% confidence interval for the log-transformed pharmacokinetic parameters AUC_0-t, AUC_0-∞ and Cmax fell within 80.00–125.00% for Oseltamivirand its metabolite. Therefore the test product of Oseltamivir tablet can be considered as bioequivalent to the reference product.

 

CONCLUSION:

Two LCMS/MS methods have been reported here for the simultaneous determination of Oseltamivir and its metabolite, Oseltamivir carboxylate using two different types of columns. Both methods have been validated as per FDA regulations. Both methods are sensitive, precise, accurate, selective and robust with no carry over or matrix effect making them suitable for routine analysis of Oseltamivir and its metabolite. The method using cation exchanger column (SCX) has distinct advantage over the reversed phase column in short run time (4.5 min vs6.0  min) which makes it ideal for high throughput analysis and suitable for industrial use. Moreover, use of isocratic elution in this ion – exchange method makes it more user friendly compared to gradient elution method used in RP chromatography. Use of ion-exchange (SCX) column is also advantageous in respect to matrix tolerance as there is only one interaction involved in the separation: the analytical species interacting with the stationary phase. The method was successfully applied to quantify simultaneously Oseltamivir and its metabolite in the bio-equivalence study. This validated method is also useful in a pharmaceutical discovery environment and may potentially be applied to therapeutic drug monitoring.

ACKNOWLEDGEMENT:

The authors thank the management of Norwich Clinical Services for providing the opportunity to complete the project.

 

CONFLICT OF INTEREST:

All authors hereby declare that no conflict of interest is associated with the publication of this manuscript.

 

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Received on 18.03.2016       Accepted on 08.04.2016     

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