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Solid phase extraction liquid chromatography mass spectrometry method with electrospray ionization for the determination of Ondansetron in human plasma: Development and validation consideration
⁎Corresponding author. Tel.: +91 9824637618. rbp.arcp@gmail.com (Rashmin Patel)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
A new liquid chromatography mass spectrometry method with electrospray ionization was developed for the estimation of Ondansetron in human plasma. Sample preparation was carried out by solid-phase extraction that enabled direct injection into the LC–MS/MS system. Ondansetron and an internal standard (ISTD), Ramosetron, were separated by using a Gemini NX C18 analytical column (100 × 4.6 mm i.d., particle size 5 μm) with isocratic elution. The mobile phase was composed of a mixture of ammonium formate buffer (pH 3.0; 2 mM) and acetonitrile (30:70, v/v), pumped at a flow rate of 0.5 mL/min. Quantitation of the analyte was performed with multiple ion monitoring (MRM) in positive ionization mode using electrospray ionization interface. The ion transitions recorded were m/z 294.3/170.0 for Ondansetron and m/z 280.3/121.3 for ISTD. The method was validated over a wide dynamic concentration range of 1.00–100.00 ng/mL (r ⩾ 0.9996). The lower limit of quantitation (LLOQ) was 1.00 ng/mL. The extraction recovery was above 81.50% for analyte and above 85.33% for ISTD. A run time of less than 3.0 min for each sample made it possible to analyze a large number of human plasma samples per day. The proposed method was successfully applied for determination of Ondansetron in bioequivalence study of 4 or 8 mg Ondansetron tablet.
Keywords
Ondansetron
Solid-phase extraction
LC–MS/MS
Human plasma
1 Introduction
Ondansetron, (RS)-9-methyl-3-[(2-methyl-1H-imidazol-1-yl)methyl]-1,2,3,9-Tetrahydro-4H-carbazol-4-one monohydrochloride, is a selective serotonin 5-HT3 receptor antagonist with antiemetic activity (Raza et al., 2007; Alam et al., 2012; Mujtaba et al., 2013). It is used in the management of nausea and vomiting induced by cytotoxic chemotherapy and radiotherapy. The antiemetic activity of the drug is brought about through the inhibition of 5-HT3 receptors present both centrally (medullary chemoreceptor zone) and peripherally (GI tract). This inhibition of 5-HT3 receptors in turn inhibits the visceral afferent stimulation of the vomiting center, likely indirectly at the level of the area postrema, as well as through direct inhibition of serotonin activity within the area postrema and the chemoreceptor trigger zone.
Literature survey revealed that Fluorescence polarization immunoassay, radioimmunoassay, HPTLC, capillary zone electrophoresis, Liquid chromatography–mass spectrometric (LC–MS/MS) method and ion-pair chromatography methods have been used to quantify Ondansetron in pharmaceutical dosage forms and biological fluids (Raza et al., 2007; Varvara et al., 2009; Alam et al., 2012; Mujtaba et al., 2013). Also, there have been reports on gradient HPLC methods with UV detection (Dedania et al., 2009; Shashela et al., 2009). However, these reported methods utilize laborious and time-consuming sample preparation procedure, gradient development, having lower sensitivity and comparatively longer run time (Koufopantelisa et al., 2009; Alvarez et al., 2011) which limit their application. In spite, of different approaches available for quatitation of ondansetron, it would be of particular interest to develop a rapid, accurate, precise and sensitive method to overcome the drawbacks of reported methods.
In view of the above facts, authors attempted the development and validation of a new, rapid, isocratic, accurate, precise and sensitive, hyphenated LC–MS/MS method for the estimation of Ondansetron in human plasma. The assay was based on 200 μL human plasma samples, following solid-phase extraction that enabled direct injection into the LC–MS/MS system using positive electrospray ionization. The method was successfully applied for the determination of Ondansetron in bioequivalence study of 4 or 8 mg Ondansetron tablet in Indian males in fasting condition.
2 Experimental
2.1 Materials
2.1.1 Reference/working standards
Ondanseron hydrochloride (purity, 99.00%) and Ramosetron hydrochloride (purity, 99.58%) were provided as gratis samples by Cadila Pharmaceuticals Limited (Ankleshwar, Gujarat, India) and Changzhou Wujin Pharmaceutical Chemical Co. Ltd (China), respectively.
2.1.2 Chemicals and reagents
Acetonitrile and Methanol were of HPLC grade and were purchased from Merck Inc. (USA). Formic acid and Ammonium formate were of AR grade and were purchased from Merck Inc. (USA). HPLC grade water was obtained in-house using a Millipore Milli-Q Gradient Water Purification System (USA).
2.1.3 Human plasma
Human plasma was obtained from the Cadila Pharmaceuticals Ltd. (CRO, dholka, India). Dipotassium ethylene diamine tetra-actetic acid (K2EDTA) was used as an anticoagulant. Haemolyzed, lipemic and Sodium-Heparin containing human plasma required for selectivity/specificity experiment were also obtained from Cadila Pharmaceuticals Ltd. (CRO, Dholka, India).
2.2 Equipment and apparatus
Analysis was performed on a LC–MS/MS system consisting of an API 4000 triple quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, USA) equipped with an electrospray ionization interface (Turbo Ion Spray) and a LC-20AD (Shimadzu, Kyoto, Japan) LC system accompanied with an autosampler SIL-HTC (Shimadzu, Kyoto, Japan). A Nitrox-N2 model UHPLC–MS12E nitrogen generator (Gateshead, England) was used to provide highly pure nitrogen that was utilized as sheath and nebulizing gas. Data acquisition and analysis were performed using Analyst Software version 1.4.2 (Applied Biosystems, USA). Micropipettes (Eppendorf India Ltd., India) of different volume capacity like, 2–20, 10–100, 20–200 and 100–1000 μL were used for transfer of sample and reagents.
2.3 Chromatographic conditions
An analytical method based on solid phase extraction has been developed for estimation of Ondansetron in human plasma using Ramosetron as an internal standard (ISTD). Lichrosep Sequence Cartridge (30 mg – 1 mL) was used for solid phase extraction. Analytes were exracted from human plasma using Positive Pressure Processor Extraction Manifold (Ezypress 48, Orochem India Pvt. Ltd. India). Analytes were separated using an analytical column, Gemini NX C18 (Phenomenex, USA), (100 mm length; 4.6 mm internal diameter; particle size 5 μm). Mobile phase was composed of ammonium formate buffer (pH 3.0 adjusted with formic acid; 2 mM) and acetonitrile (30:70, v/v). The aliquots of resulting samples were analyzed by combining liquid chromatography and tandem mass spectrometry (LC–MS/MS) with positive ion Electro spray ionization using multiple reaction monitoring (MRM). The elution was achieved isocratically at a flow rate of 0.5 mL/min with injection volume of 5 μL. The run time was as low as 3.0 min.
2.4 Mass spectrometric conditions
The analytes were detected by an API 4000 triple quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, USA) equipped with an electrospray ionization interface (Turbo Ion Spray). The Analyst Software version 1.4.2 (Applied Biosystems) was used for system control, data acquisition and quantification. The ESI source was working in the positive ionization mode, and an ion-spray voltage of +5500 V was applied. The capillary temperature was set to 400 °C. The system was tuned using a continuous 5 μL/min infusion of an Ondansetron (200 ng/mL) solution in acetonitrile. The signal was optimized on the total ion current in MS mode. The protonated precursor molecular ions [MH]+ of Ondansetron (m/z 294.3) and ISTD (m/z 280.3) were trapped with a mass resolution of 1.0 amu and fragmented by collision induced dissociation with an activation time of 200 ms; collision energy of 32 and 34 V, respectively; collision gas pressure, 7 psi; curtain gas pressure, 30 psi; nebulizer gas pressure 40 psi; heater gas pressure 60 psi. The daughter ions resulting from these fragmentations were monitored in multiple reaction monitoring (MRM). Ondansetron was identified by the presence of daughters’ ions at m/z 170.0 and ISTD was identified by the presence of daughter ions at m/z 121.3.
2.5 Preparation of calibration curve standards (CC) and quality control (QC) samples
Ondansetron stock solution (1000.00 ng/mL) and intermediate stock solution (10.00 ng/mL) for CC samples were prepared in methanol–water (50:50, v/v). ISTD stock solution (1000.00 ng/mL) and intermediate ISTD dilution (500.00 ng/mL) were prepared in methanol–water (50:50, v/v). CC samples were prepared by spiking respective intermediate stock solutions in blank human plasma at concentrations of 1, 2, 6, 15, 30, 50, 80 and 100 ng/mL. CC samples were prepared from a screened and pooled K2EDTA containing human blank plasma. Ondansetron stock solution for QC was prepared separately and QC plasma samples were prepared at 1, 3, 14, 35, and 70 ng/mL (LLOQ QC, LQC, MQC2, MQC1 and HQC, repectively) in the same manner as for plasma standard. QC samples were prepared from different matrix pools on each day of analysis. Zero standard was prepared by diluting 20 μL of ISTD dilution (500 ng/mL) with 200 μL of screened plasma. All prepared plasma samples were stored at temperature of −20 ± 5 °C or −70 ± 5 °C in deep freezer (Sanyo, Germany) and all prepared stock solutions were stored at 2–8 °C in refrigerator (Vestfrost, UK).
2.6 Preparation of working solutions for short term/long term stock solution stability
Working spiking solutions for short term/long term stock solution stability were prepared by diluting drug intermediate stock solution (10.000 μg/mL) with diluent (methanol–water, 60:40, v/v).
2.7 Sample preparation
2.7.1 Extracted sample preparation
The required number of samples from the deep freezer were retrieved and thawed in water bath maintained at room temperature and the tubes were vortexed. 0.2 mL of sample was transferred into a pre-labeled tube. 20 μL of ISTD dilution (0.500 μg/mL) was added to all the samples except plasma blank (STD BL) and vortexed. 20 μL diluent (methanol–water, 60:40, v/v) was added in STD BL sample and vortex mixed. The conditioning of cartridges (Lichrosep Sequence 30 mg – 1 mL) was carried out with 1 mL methanol followed by 1 mL Milli-Q water. CC standard or QC samples were loaded in cartridge respectively. Cartridges were washed with 1 mL Milli-Q water twice. Cartridges were dried at maximum pressure in Positive Pressure Processor for 2 min. All the samples were eluted with 1 mL mobile phase. About 0.6 mL of eluted solution was transferred in pre-labeled autosampler vials and were arranged in the auto-sampler and injected in LC–MS/MS system.
2.7.2 Aqueous sample preparation
Aqueous samples were prepared by transferring 2440 μL of mobile phase in prelabelled tubes. 10 μL of respective spiking solutions was added in prelabelled tubes and was vortexed. 50 μL of ISTD dilution (0.500 μg/mL) was added and vortex-mixed. The samples were transferred into prelabelled auto-sampler vials and injected in LC–MS/MS system.
2.8 Validation
The method was validated for accuracy, precision, sensitivity/specificity, calibration curve range, and reproducibility according to the USFDA guideline for validation of bioanalytical methods (Guidance for the industry: Bioanalytical method validation, 2001).
2.8.1 Calibration curve and linearity
An eight-point calibration curve was constructed by plotting peak area ratio (y) of Ondansetron to the ISTD versus Ondansetron concentrations (x). Analysis of CC samples at each concentration was performed in duplicate. Results for blank samples were not used as part of the calibration curve. Slope, intercept and correlation coefficient were calculated as regression parameters by weighted (1/x2) linear regression.
2.8.2 Intra-day and inter-day precision and accuracy
The following samples plasma blank, zero standard, calibration standards, QC samples (six replicates) were injected in a sequence for a precision and accuracy batch. Samples were processed and the concentrations of quality control samples were calculated by quantifying them against calibration standards. One precision and accuracy batch on single day was generated for intra-day precision and accuracy. Three precision and accuracy batches were generated on different days for inter-day precision and accuracy.
2.8.3 Specificity
Samples of six normal matrices, one lipemic and one haemolyzed matrix were obtained. The lowest calibration standard sample and blank sample in each matrix were prepared. The interference in each blank sample was checked by comparing the area response obtained in the lowest calibration sample of that matrix using an established procedure for analysis. % interference was calculated.
2.8.4 Matrix effect
Three samples each of LQC and HQC were prepared from each of six blank plasmas. These QC samples along with calibration standards prepared from one blank matrix were injected and these QC samples were quantified against the calibration curve to calculate concentration.
2.8.5 Recovery
The recovery of Ondansetron and internal standard (Ramosetron) by comparing the bioanalytical results for extracted QC samples with aqueous solutions equivalent to 100% recovery of LQC, MQC and HQC was evaluated. Six samples each of LQC, MQC and HQC (six replicates) were processed. The comparison samples in aqueous medium at low, medium and high quality control level were prepared which constitute spiking of same concentration levels in a similar volume as that of extracted samples. These comparison samples were analyzed along with extracted QC samples and% mean recovery was calculated by comparing area obtained in extracted samples with that of aqueous samples. The % mean recovery of internal standard was calculated at all levels.
2.8.6 Dilution integrity
Aqueous solution having concentration approximately three times of ULOQ was prepared. Aqueous sample was spiked in blank plasma. Then spiked matrix was diluted five and ten times with blank plasma. Six samples each from five times and ten times diluted matrix were processed and injected along with calibration standards and these diluted QC smaples were quantified against calibration curve to calculate concentration.
2.8.7 Stability
The short term stability of Ondansetron and ISTD in stock solutions was carried out for 6 h from the time of preparation. The long term stability of Ondansetron and ISTD in stock solutions was carried out for 6 days from the time of preparation. The % mean stability was calculated. Stability of Ondansetron in human plasma was carried out at low and high quality control levels (six replicates). Freeze–thaw stability was carried out using stability samples at −70 °C and five freeze–thaw cycles. Bench top stability was carried out by keeping stability samples on bench at room temperature for 13 h 20 min. Wet extract stability was carried out by processing stability samples and stored in a wet extract form at 5 °C in a refrigerator for 24 h. Autosampler stability was carried out after stability samples were stored in auto sampler for 52 h at 5 °C. The % change was calculated.
3 Results and discussion
3.1 Optimized chromatographic conditions
To separate the Ondansetron (analyte) and Ramosetron (ISTD) three types of stationary phase columns were tried including Hipurity C18 (50 × 4.6 mm, 5 μm), Phenomenex C18 (100 × 4.6 mm, 5 μm) and Gemini NX C18 (100 × 4.6 mm, 5 μm). Poor chromatography in terms of resolution and symmetric factor for Ondansetron was observed on Hipurity C18 (100 × 4.6 mm, 5 μm) and Phenomenex C18 (100 × 4.6 mm, 5 μm) columns although various percentages of acetonitrile and different pH of buffers were tested. Ondansetron was extracted using different types of extraction methods like precipitation and solid phase extraction from the spiked plasma samples. Response of blank plasma was observed in the precipitation extraction method. Then solid phase extraction was carried out for extraction of Ondansetron using Lichrosep Sequence Cartridge (30 mg – 1 mL). Interference in blank plasma response was resolved in the solid phase extraction method. In order to make the mobile phase suitable for LC–MS/MS, the probability of using higher proportion of organic component of acetonitrile was attempted. After achieving good chromatography in Gemini NX C18 (100 × 4.6 mm, 5 μm), the saturation of ISTD response was observed but it was resolved by diluting the ISTD dilution to appropriate concentration. Representative chromatograms of (A) human plasma blank (B) Zero Standard (C) Ondansetron and (D) Ramosetron are shown in Fig. 1.
Finally, all the subsequent plasma samples thus run with the mobile phase consisted of ammonium formate buffer (pH 3.0, adjusted with formic acid; 2 mM) – acetonitrile (30:70, v/v) and were separated using an analytical column, Gemini NX C18 (100 × 4.6 mm, 5 μm). The elution was achieved isocratically at a flow rate of 0.5 mL/min with injection volume of 5 μL.
3.2 Method validation
The calibration curve (Fig. 2) was linear over the concentration range of 1.00–100.00 ng/mL of Ondansetron in human plasma with a regression coefficient of determination (r) ⩾ 0.9990. The average slope and intercept of regression equations were 0.120 and 0.003, respectively. The calibration curve parameters are summarized in Table 1. Linearity was found to be quite satisfactory and reproducible with time.
| P&A batch ID | Slope | Intercept | r |
|---|---|---|---|
| P&A I | 0.119 | 0.015 | 0.9996 |
| P&A III | 0.120 | 0.003 | 0.9990 |
| P&A IV | 0.120 | 0.002 | 0.9992 |
The precision and accuracy data for QCs are summarized in Table 2. For QC samples at 1.00 ng/mL (LLOQ), intra-assay mean accuracy and precision values were 90.73% and 4.18%, respectively, while above LLOQ they were 96.53–104.25% and ⩽3.00%. Inter-assay mean accuracy and precision at LLOQ were 98.61% and 7.76%, respectively and above LLOQ, 95.93–100.11% and ⩽4.02%, respectively.
| QC sample ID | HQC | MQC1 | MQC2 | LQC | LLOQ QC |
|---|---|---|---|---|---|
| Nominal concentration (ng/mL) | 70.000 | 35.000 | 14.000 | 3.000 | 1.000 |
| Back calculated concentration (ng/mL) | |||||
| Intra-day (n = 6) | |||||
| Mean | 72.897 | 35.710 | 14.595 | 2.896 | 0.907 |
| SD | 2.186 | 0.593 | 0.124 | 0.056 | 0.038 |
| % CV | 3.000 | 1.660 | 0.850 | 1.940 | 4.180 |
| % Mean accuracy | 104.140 | 102.030 | 104.250 | 96.530 | 90.730 |
| Inter-day (n = 18) | |||||
| Mean | 69.746 | 34.122 | 14.016 | 2.878 | 0.986 |
| SD | 2.800 | 1.273 | 0.502 | 0.083 | 0.077 |
| % CV | 4.020 | 3.730 | 3.580 | 2.890 | 7.760 |
| % Mean accuracy | 99.640 | 97.490 | 100.110 | 95.930 | 98.611 |
The specificity of the analytical method was investigated by analyzing six individual human blank plasma samples. The acceptance criteria for Ondansetron were mean interference per batch ⩽20% of signal at LLOQ, and that for ISTD was mean interference per batch ⩽5% of signal at working concentration. No interference peak was detected for Ondansetron or ISTD.
Three samples of each LLOQ from each seven plasma lots were prepared and injected along with calibration standards prepared from one blank plasma. The % accuracy of LLOQ samples prepared with the different biological matrix lots was found to be 91.88%, which is within the range of 80.00–120.00% and precision (% CV) was found to be 3.42% which is acceptance criteria of 20.00%. All of the matrix lots were found to be within the acceptance criteria (at least 80% of samples).
The extraction recovery was assessed by comparison of the responses obtained from analysis of extracted spiked human samples and unextracted samples. The absolute extraction recoveries for Ondansetron and ISTD were 81.50% and 85.33%, respectively (Table 3).
| Replicate No. | HQC | MQC1 | MQC2 | LQC | ||||
|---|---|---|---|---|---|---|---|---|
| Extracted peak area | Un-extracted peak area | Extracted peak area | Un-extracted peak area | Extracted peak area | Un-extracted peak area | Extracted peak area | Un-extracted peak area | |
| Mean | 2,039,854 | 2,454,094 | 1,002,701 | 1,262,515 | 404,234 | 495,887 | 88,293 | 107,908 |
| SD | 15,191 | 66,286 | 61,745 | 6429 | 3564 | 3818 | 926 | 2621 |
| % CV | 0.740 | 2.700 | 0.620 | 0.510 | 0.880 | 0.770 | 1.050 | 2.430 |
| % Mean recovery | 83.120 | 79.420 | 81.520 | 81.820 | ||||
| % Overall recovery | 81.500 | |||||||
The% mean accuracy of the DI 1/2 samples was found to be 107.350% with dilution factor of 2. The % mean accuracy of the DI 1/10 samples was found to be 104.810% with dilution factor of 10% CV of DI 1/2 samples and DI 1/10 samples was found to be 5.090% and 3.950% (Table 4).
| Replicate No. | DI spiked standard concentration (ng/mL) | |||
|---|---|---|---|---|
| DI 1/2 sample (ng/mL) | DI 1/10 sample (ng/mL) | |||
| 85.000 | 170.000 | 17.000 | 170.000 | |
| Without DF | With DF | Without DF | With DF | |
| Mean | 91.251 | 182.501 | 17.818 | 178.177 |
| SD | 04.647 | 09.294 | 00.703 | 07.034 |
| % CV | 05.090 | 05.090 | 03.950 | 03.950 |
| % Mean accuracy | 107.350 | 107.350 | 104.810 | 104.810 |
% Mean short term stock solution stability for drug and ISTD was found to be 102.650% and 108.670%, respectively. The short term stability of Ondansetron and ISTD in stock solutions was found to be 7 h 10 min at ambient temperature for both. The % mean long term stock solution stability for drug and ISTD was found to be 99.540% and 99.860%, respectively. The long term stability of Ondansetron and ISTD in stock solutions was found to be 06 days at 5 ± 3 °C. The % mean stability for freeze–thaw stability for drug and ISTD was found to be 99.540% and 99.860%, respectively. Freeze–thaw stability samples were found to be stable for five freeze–thaw cycles at −70 ± 5 °C. Bench top stability was found to be 13 h 20 min at ambient temperature. Wet extract stability was found to be 43 h 30 min at 5 ± 3 °C. Autosampler stability was found to be 56 h 46 min at 5 ± 3 °C (Table 5).
| Stability | Nominal area observed | Mean area observed | % Mean stability |
|---|---|---|---|
| Short term stock solution stability | |||
| Ondansetron | 3,925,802 | 4,029,915 | 102.650 |
| ISTD | 367,649 | 399,512 | 108.670 |
| Long term stock solution stability | |||
| Ondansetron | 9,685,504 | 9,641,251 | 99.540 |
| ISTD | 682,654 | 681,732 | 99.860 |
4 Conclusion
The developed rapid Hyphenated Liquid Chromatography-Tandem Mass Spectrometry method using API-4000 mass spectrometer substantially improves sensitivity, accuracy and precision for the quantitation of Ondansetron in human plasma at a concentration range of 1.00–100.00 ng/mL. The proposed method enabled the reliable determination of Ondansetron in bioequivalence study of 4 or 8 mg Ondansetron tablet. Selectivity and sensitivity were sufficient for detecting and quantifying Ondansetron in human plasma. These features coupled with a short run time at 3.0 min compared to reported methods which allow high sample throughput with almost 500 samples per day. The proposed method enabled the reliable determination of Ondansetron in bioequivalence study of 4 or 8 mg Ondansetron tablet.
References
- Int. Res. J. Pharm.. 2012;3:111-113.
- J. Chromatogr. B. 2011;879:186-190.
- Asian J. Res. Chem.. 2009;2:108-111.
- Guidance for the industry: Bioanalytical method validation, 2001. Department of health and human services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Centre for Veterinary Medicine (CVM), Washington DC, US. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdf dated 30.06.2013.
- J. Chromatogr. B. 2009;877:3850-3856.
- J. Drug Testing Anal.. 2013;5:122-125.
- J. Chin. Chem. Soc.. 2007;54:223-227.
- Chromatographia. 2009;70:75-81.
- Farmac. 2009;57:442-451.