|Year : 2013 | Volume
| Issue : 1 | Page : 74-79
Development and validation of an improved LC-MS/MS method for the quantification of desloratadine and its metabolite in human plasma using deutrated desloratadine as internal standard
M Saquib Hasnain1, Shireen Rao2, Manoj Kr. Singh2, Nitin Vig2, Manish Kr. Singh2, Subodh Kr. Budakoti2, Abdulla Ansari2
1 Department of Pharmaceutical Chemistry, Seemanta Institute of Pharmaceutical Sciences, Orissa, India
2 Bioanalytical Division, Fortis Clinical Research Limited, Faridabad, Haryana, India
|Date of Submission||16-Jun-2012|
|Date of Decision||19-Jul-2012|
|Date of Acceptance||01-Aug-2012|
|Date of Web Publication||28-Jan-2013|
M Saquib Hasnain
Department of Pharmaceutical Chemistry, Seemanta Institute of Pharmaceutical Sciences, Orissa
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Purpose: For the determination of desloratadine (DES) and 3-OH desloratadine (3-OHD) in human plasma using deutrated desloratadine (DESD5) as internal standard (IS), a novel stability indicating liquid chromatography-tandem mass spectrometric method was developed and validated to support the clinical advancement. Materials and Methods: The solid-phase extraction method used for sample preparation and calibration range was 100-11,000 pg/ml, for which a quadratic regression (1/x 2 ) was best fitted. The blank plasma was screened and observed free from any endogenous interference. Results: The accuracy (% nominal) at low limit of quantification LLOQ level for DES and 3-OHD was 100.4% and 99.9% whereas precision (%CV) was 4.6 and 5.1%. They (DES and 3-OHD) were stable in human plasma after five freeze-thaw cycles, at room temperature for 23.8 hour, bench top stability for 6.4 hour. Conclusion: This method fulfills all the regulatory requirements for selectivity, sensitivity, precision, accuracy, stability, goodness of fit, and ruggedness of the method for the determination of DES and 3-OHD in human plasma.
Keywords: Desloratadine, method validation, 3-OH desloratadine
|How to cite this article:|
Hasnain M S, Rao S, Singh MK, Vig N, Singh MK, Budakoti SK, Ansari A. Development and validation of an improved LC-MS/MS method for the quantification of desloratadine and its metabolite in human plasma using deutrated desloratadine as internal standard. J Pharm Bioall Sci 2013;5:74-9
|How to cite this URL:|
Hasnain M S, Rao S, Singh MK, Vig N, Singh MK, Budakoti SK, Ansari A. Development and validation of an improved LC-MS/MS method for the quantification of desloratadine and its metabolite in human plasma using deutrated desloratadine as internal standard. J Pharm Bioall Sci [serial online] 2013 [cited 2020 Feb 21];5:74-9. Available from: http://www.jpbsonline.org/text.asp?2013/5/1/74/106571
Desloratadine (DES), a major metabolite of loratadine also known as descarboethoxyloratadine and its chemical name is 8-chloro-6,11-dihydro-(4-piperdinylidene)-5H-benzo [5,6]cycloheptal [1,2-b] pyridine which is a tricyclic anti-histamine, orally active non-sedating drug, indicated for the relief of the nasal and non-nasal symptoms of seasonal allergic rhinitis in patient more than two years of age.  As compared with loratadine, it shows a higher affinity for H1 human receptor. DES is instable to light and heat, having poor solubility. There are several method for the analysis of DES and its metabolites, such as ultra violet detection,  mass spectrometry detection,  liquid chromatography with fluorescence detection,  gas chromatography with nitrogen-phosphorus detection,  etc., were available but usually, bio analysis of DES and its metabolites in biological matrix are performed with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Following methods such as protein precipitation, liquid-liquid extraction or solid-phase extraction with robotic liquid handling systems are used to prepare the sample for bioanalysis. ,,,,,, The detector used mainly is a triple quadrupole mass spectrometer equipped with either an electrospray or an atmospheric pressure chemical ionization ion source. DES is a selective, potent, peripheral H1 receptor antagonist, which is orally active whereas 3-OH desloratadine (3-OHD) is a major active metabolite of both DES and loratadine. For the determination of DES and 3-OHD in human plasma, a solid-phase extraction LC-MS-MS method is developed with an LLOQ of 100 pg/ml, which is further validated to the great extent in our bio-analytical laboratories. Previously, Xu, et al. developed the method but that method has longer retention time and less recovery as compared with our developed method. This method has just half retention time and recovery double of Xu, et al.  The stability of these analyses in human plasma under the most favorable condition is validated as part of the validation. This method was fulfilling the acceptance criteria for bio-analytical method validation as per ICH guidelines.
| Materials and Methods|| |
Test compounds and internal standards (IS): DES was purchased from Divis Laboratories, 3-OHD from Vardha Biotech Ltd. and DES-D5 (deutrated DES) from Vivan Life Sciences of India.
For calibration standards (CC) and quality control (QC) sample preparation, human plasma with K2 EDTA is used. Human plasma with K2 EDTA was purchased from Yash Laboratories (Faridabad, Haryana, India) and chromatographically screened for interfering substances. For this blank plasma was run as part of each validation to overcome the interference.
Acetonitrile HPLC grade from J. T Baker, disodium hydrogen phosphate AR grade from Merck Ltd (Mumbai, India), ammonia AR grade, formic acid AR grade, methanol HPLC grade, ortho-phosphoric acid AR grade, acetone HPLC grade from Rankem (RFCL Ltd, New Delhi), ammonium formate (CDH, New Delhi), and water HPLC grade were used.
Five-millimolar disodium hydrogen phosphate and 10 mM ammonium formate solution were prepared appropriately. Mobile phase was prepared by adding solution A and B in a ratio of 90:10, where solution A was acetonitrile and methanol in a ratio of 40:60 and solution B was methanol and water in a ratio of 90:10. Diluent solution was 50:50 methanol and water. Eluent solution was prepared by mixing ammonia and methanol in a ratio of 3:97.
Stock solution of 3-OHD and DES was prepared in the diluents solution having 1mg/ml of strength. These stock solutions were used to prepare spiking solution of the above-working standards. From this stock solution, a stock dilution of 50 ng was prepared for spiking of calibration standard (CC) and quality control (QC) standards of 3-OHD and DES. The CC and QC samples were used to evaluate the accuracy and precision. It was also used for the determination of low limit of quantification (LLOQ). Stock solution of DES-D5 was used as an IS working solution. The stock solutions of these compounds were screened chromatographically and by mass spectrometry for interfering elements.
Two drugs 3-OHD and DES were analyzed using a AB SCIEX API-4000 triple quadrupole mass spectrometer and HPLC used was schimadzu. The analytical column used in this method was hypurity Advance 50 × 4.6 mm, 5-μm particle size. Mobile phase used was a mixture of solution A and B in a ratio of 90:10 and the flow rate was maintained at 1 ml/min. Fifteen-microliter injection volumes and 5 min was the run time. By applying the above condition, the respective retention time of 3-OHD, DES, and DES-D5 (IS) was reported as 0.52 min. The mass spectrometer was used in positive ion mode having turbo ion spray ionization. The following parameters were set for mass spectrometer: curtain gas on 25, gas source-1 on 50, gas source-2 on 40, ion spray voltage on 3000 V, temperature on 500°C collision gas on seven and entrance potential was set 10 V, respectively. The dwell time was 200 msec. A summary of mass condition was shown in [Table 1]. The multiple reaction monitoring (MRM) conditions were monitored for the analytes and IS: for DES, 3-OHD and DES-D5, it was 311.03/259.10, 326.97/274.97, and for 316.02/264.20, respectively. Data were analyzed by using Analyst 1.5 Software.
Calibration standards and quality control samples
The spiked plasma with 3-OHD and DES was used for CC and QC samples. The range of the required CC and QC sample was 100-11200 pg. A set of nine CC standards of pooled plasma was prepared and stored at −22°C. The QC samples were stored in the same environment. In validation each run consist of CC and six replicates of QC sample at low (0.284), medium (4.5), and high (9.0) ng/ml level, respectively. The lower limit of quantification (0.100 ng/ml) was analyzed to determine the accuracy and precision.
The validation of method was assessed by three analytical runs and each of them includes nine CC with QC samples. Two standards zero (with IS) and blank (without IS) were also incorporated in the runs. At least two-thirds of CC standards must pass individually. The individual accuracy for LLOQ must be within ± 15% of nominal ± 20%. Among three analytical runs, at least one of the two CC standards at LLOQ and ULOQ level must be passed. The CC coefficient of determinant (r 2 ) must be greater than 0.98. Within-run and between-run accuracy must be within ± 15% at QC levels and individual accuracy at each concentration must be within ± 15%.
Method selectivity was assessed by screening 20 blank plasma batches to check the interference at the retention time of analytes and IS. For acceptance, it must be less than 20% of the mean peak response calculated from the analysis of LLOQ QC samples at the expected retention time of analytes and less than 5% of IS.
Matrix effect is the suppression or enhancement of ionization of analytes by the presence of matrix in the biological samples and quantitative measure of the matrix effect termed as matrix factor. For this spiked analytes and IS at LQC and HQC, QC level into each of blank plasma extracts from six different batches of matrix, respectively, and analyzed in duplicate along with six replicates of aqueous sample at same QC level. The variability of matrix factor was measured as coefficient of variation and must be less than 15%.
The dilution integrity experiment was performed to determine the accurate quantitation within the range of CC at a concentration greater than the ULOQ, which can be diluted with the matrix. The individual and mean accuracy must be less than ± 15%. Individually two-third dilution QC samples must meet the above criteria.
The stability was checked by analyzing the QC samples of analytes at lower and higher concentration (n = 6) that were stored under various condition. For the acceptance, two-third stability QC samples must have individual accuracy within ± 15% and mean accuracy should be ± 15%. The stability of the analytes in stored stock solution during long-term stability for 44 days was determined by comparing it with the fresh stock solution. It should be pass if the mean difference between the two was less than 5%.
The stability of solution under different condition likes at room temperature, at refrigerator temperature for 6, 16 and 43 days was demonstrated during analysis. The reference mix stability at 24 hours and reinjection reproducibility was done.
For the evaluation of ruggedness of the method, run one partial accuracy batch using a different column (same type) by a different analyst employing the same or different instrument.
Withdraw required number of samples from the sample storage device and allowed them to thaw at room temperature. Vortex the thawed samples to ensure complete mixing of contents. 50 μl of IS dilution were aliquot (50 ng/ml of DES-D5) into micro centrifuge tubes and added 500 μl of each sample, Vortex it to mix well and add 400 μl of 10 Mm of disodium hydrogen phosphate solution and again vortex it. Place required number of oasis HLB cartridges in positive pressure solid-phase extraction assembly for sample preparation. Condition the cartridges with 1 ml of methanol followed by 1ml of water. Samples were loaded into the cartridges and passed through the cartridges under constant pressure. Then it is washed with 1ml of 10 mM disodium hydrogen phosphate solution followed by 1ml of water twice. Then elute them with 1 ml of elution solution under constant pressure, and dried on nitrogen evaporator at 50°C and 20 psi until complete drying. Reconstituted with 400 μl of mobile phase and transferred in the micro centrifuge tubes. Spin at 15000 rpm and 5°C for 2 minutes; transfer the supernatant in cleaned auto sampler vials.
| Results and Discussion|| |
For the sample extraction, solid-phase extraction was selected due to following reasons: First to minimize the matrix effect by a clean extract by which we could obtain the appropriate sensitivity at LLOQ level, i.e., 0.100 ng/ml and lastly it was very useful to achieve large clinical application for pharmacokinetic study. All the available extraction procedure was considered during extraction procedure, but amongst them solid-phase extraction gave brilliant result. Matrix effect was initially found and reduced up to 80% by using this method. Hence it was considered as the best procedure for the extraction.
Samples were eluted using ammonia and methanol solution in a ratio of 3:97, yields a very good recovery of 74.6% for DES and 69.3% for 3-OHD. Chromatographic peak shapes were symmetric, recovery found to be consistent and highly sensitive for all the analytes and IS at the LLOQ level.
Tandem mass spectrometry
The product ion mass spectra and scanning of DES, 3-OHD and DES-D5 were obtained using positive ion mode. The scanned mass spectra of DES, 3-OHD and DES-D5 were signals for the protonated molecular ions at m/z = 311.03, m/z = 326.97 and 316.02, respectively. Product ion mass spectra for above compound were 259.10, 274.97, and 264.20. Transition from m/z 311.03 to m/z 259.1, m/z 326.97 to m/z 274.97, and m/z 316.02 to 264.20 were monitored in MRM mode for the quantification.
In preclinical volunteers, hydroxy metabolites of 3-OHD at different position were present and these metabolites have same molecular masses and almost same product ion spectra, due to which there separation were difficult. To overcome this problem, a hypurity advance 50 × 4.6 mm, 5 μ column and a flow rate of 1 ml/min with a suitable post-column splitter were used.
Sensitivity of the method was determined in one of the validation runs at LLOQ (0.100 ng/ml) level by processing six replicates of it for DES and 3-OHD. The batch precision and accuracy at LLOQ level using IS ratio method was 4.6% and 100.4% for DES while 5.1% and 99.9% for 3-OHD. The representative chromatogram of a calibration standard at the LLOQ level is shown in [Figure 1]a-c.
Matrix effect is the suppression or enhancement of ionization of analytes by the presence of matrix in the biological samples. Initially it was found that matrix effect was too high. Hence the method was so designed that it reduces the matrix effect significantly. By applying this method for validation of DES, 3-OHD and DES-D5, matrix effect was lowered up to 80%. The %CV of matrix effect at LQC level for DES and 3-OHD were recorded as 2 and 2.2%, while at HQC level 0.9 and 1.4%. The above reported values show that matrix effect of plasma was significantly reduced. Blank chromatograms of these components are shown in [Figure 2]a-c.
Selectivity was done by screening 20 different batches of blank plasma which did not contain any interference plasma components or other sources at the retention time of analytes and IS. There was not any interference reported at the retention time of analytes and IS.
Calibration curve regression
Calibration curve regression for both DES and 3-OHD was a quadratic regression (1/concentration) which gave the best fit and coefficient of determination r 2 for validation were greater than 0.998.
Accuracy and precision
The accuracy (% nominal) for DES within batch and between batches was reported as 100.4 and 100.1% while %CV within batch and between batches was reported as 4.6 and 4.4%, respectively. Similarly accuracy (% nominal) for 3-OHD, within batch and between batch was reported as 99.9 and 100%, respectively. The precision (%CV) within batch and between batches was 5.1 and 5%. The accuracy and precision of DES and 3-OHD were summarized in [Table 2].
Recoveries were determined by comparing the peak area of the QC sample before extraction to the peak area after extraction. Recovery of DES at low, medium and high QC level were 76, 74.2, and 73.6% simultaneously recovery of 3-OHD were 70.4, 69.6, and 68.1%. The % recovery (mean) for DES, 3-OHD, and DES-D5 was 74.6, 69.3, and 72.9%, respectively.
Integrity of dilution
Dilution integrity quality control (DIQC) samples were prepared and diluted two and four times with blank matrix. DES and 3-OHD accuracy values for dilution integrity were found to be 97.9 and 97.4% for DES and 95.1 and 97.1% for 3-OHD at two and four times dilution. While precision values were 1.3 and 1.8% for DES and 1.9 and 7.8% for 3-OHD.
It was assessed to determine that whether analytes and IS were stable under different storage and processing condition. The time required for sample processing was not too short to overawed the instability issues. Hence analytes stability sample transport, storage and preparation were concerned. It was done using QC samples (n = 6) at LQC, MQC and HQC level and assessed by comparing the mean value of the stability QC sample at each level with mean value of same QC pooled freshly. It was found that DES and 3-OHD were stable in human plasma. The obtained values of different stability studies for DES and 3-OHD were summarized in the table like freeze thaw stability, bench top stability, bench top extraction stability, in-injector stability [Table 3]. Stock solution stability at room temperature (in hours), at refrigerator temperature (6 and 16 days), reference mix stability (23.88 hours), long-term stability using K2EDTA below-15°C (11 days) were shown in [Table 3]. From [Table 3], it was clear that stability samples of the analytes and IS were stable at various stability condition listed above.
For the evaluation of ruggedness of the method, run one precision and accuracy batch using a different column (same type) by a different analyst employing the same or different instrument and was recorded as within batch accuracy (% nominal) for DES and 3-OHD were 97.7% and 101.7% while for within batch precision (%CV) 3.7% and 4.8%, respectively. The values reported above for DES and 3-OHD indicates that method may be used roughly at different condition.
| Conclusions|| |
The proposed method is simple, specific, accurate, linear and validated for the determination of DES and 3-OHD in human plasma over a range of 100-11,000 pg/ml. This offers a rapid and simple sample preparation which facilitates the bio-study of DES and 3-OHD, method is stability indicating and it can be used for the routine analysis of samples. The results obtained from the validation of method were satisfactory. The regulatory requirements for accuracy, precision, sensitivity, selectivity, stability and ruggedness were followed and achieved.
| Acknowledgment|| |
The authors are very thankful to the management of Fortis clinical research limited, Faridabad for supporting this work.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]
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