DEVELOPMENT AND VALIDATION OF BIOANALYTICAL METHOD FOR
ESTIMATION OF MOZAVAPTAN IN HUMAN PLASMA USING LC-MS/MS
PATEL ANKUR P.*1, AKHTAR JAWED1
1. Department of Quality Assurance, School of Pharmaceutical Sciences, Jaipur National University, Jagatpura, Jaipur-302025, Rajasthan, India.
ABSTRACT
A simple, accurate liquid chromatography with tandem mass spectrometry (LC/MS-MS) method has been developed and validated in human plasma. The method employed liquid-liquid extraction. Samples containing Mozavaptan were chromatographed on a Intersil ODS-2 column (5μm, 150 x 4.6 mm) at a temperature of 40°C.
The isocratic mobile phase composition was a mixture of Methanol / 0.1% formic acid), which was pumped at a flow rate of 1.0 mL / min with split ratio of 90:10.The retention time under these chromatographic conditions was found to be 1.0 minute with run time 2.6 minute. Diethyl ether was found to be good extracting and produced a satisfactory chromatogram. The developed LC/MS-MS method was found to be selective, simple, sensitive, accurate and linear for the analysis of Mozavaptan in human plasma. The retention time and inturn run time was very short, hence required less mobile phase for the method, making it more economical and rapid.
The method was applicable for the pharmacokinetic study of Mozavaptan.
Key words:Mozavaptan, LC/MS-MS, Validation, Plasma.
INTRODUCTION
Mozavaptan is a vasopressin receptor antagonist, chemically described as N-[4-(5- Dimethylamino-2,3,4,5- tetrahydro-1-benzazepine-1-carbonyl)phenyl]-2-methylbenzamide (fig. 1), used for treatment of hyponatremia (low blood sodium levels) caused by syndrome of inappropriate antidiuretic hormone (SIADH) due to ADH producing tumors. Mozavaptan is off white powder, freely soluble in methanol.[3,4,5,6] Literature review does not reveal any kind of bioanalytical method for estimation of Mozavaptan in human plasma.[7,8,9] The present study was designed to develop and validate a new LC/MS-MS method for the analysis of Mozavaptan in human plasma. This paper described a newly developed LC–MS/MS method for the quantitation of Mozavaptan in human plasma. The method was validated in compliance with ICH and FDA guidelines. [1, 2]
MATERIALS AND METHODS:
Mozavaptan was provided as a gift sample by Zyfine Ltd, Ahmedabad, India and its claimed purity was 99.3%.
Cilostazole (internal standard) was provided as a gift sample by Cadila Healthcare Ltd, Ahmedabad, India and its claimed purity was 98.9%. Diethyl ether (HPLC Grade), Ethyl acetate (HPLC Grade), Methyl Tert Butyl Ether (HPLC Grade) Formic acid (GR Grade) from Merck (India), Acetonitrile (HPLC Grade) and methanol (HPLC Grade) from Spectrochem. A Milli-Q system (Millipore, Bedford, MA, USA) was used.
The system Sciex API 4000 (Applied Biosystems, Canada) is equipped with LC-20AD, Prominance, pump, Shimadzu, combined with a SIL-HTc autosampler, and an DGU-20A3 vacuum degasser (Japan). The chromatography was performed on a Intersil ODS-2(5µm, 150 x 4.6 mm) at a temperature of 40°C. The isocratic mobile phase composition was a mixture of methanol: 0.1% Formic Acid, which was pumped at a flow rate of 1.0 mL / min with split ratio of 90:10. Mass spectrometric detection was performed on a triple quadrapole instrument using multiple reaction monitoring (MRM). A turbo electrospray interface in positive ionization mode was used. Data processing was performed using Analyst software version 1.4.2.
Preparation of standard solution
A stock solution was prepared by dissolving accurately weighed quantity of Mozavaptan in methanol to yield a final concentration of 1mg /mL, sonicated for 5 minutes, allowed to equilibrate to room temperature and suitably diluted with methanol. The stock solution was further diluted by suitable dilution with methanol.
Extraction of Mozavaptan from plasma
A 100 µL volume of plasma was transferred to a 4mL ria vial, and then 50 µL of IS working solution (500 ng/mL) was spiked. After vortexing for 30 sec, 3 ml of extraction solvent Diethyl ether was added. The sample was vortex-mixed for 2 min and then centrifuged at 4000 rpm for 5 minutes at 10°C. The organic layer (2.0 mL) was quantitatively transferred to a 4 mL glass tube and evaporated to dryness at 40°C under a stream of nitrogen. Then, the dried extract was reconstituted in 100 µL of Mobile phase and a 5 µL aliquot was injected into the chromatographic system.
Preparation of calibration curve and Quality control samples
Standard stock solution of Mozavaptan (1.0 mg/mL) and Cilostazole ISTD (1.0 mg/mL) were separately prepared in methanol. Spiking solutions for calibration curve and quality controls were prepared by appropriate dilution in methanol:water (50:50). Analyte (200 µg/mL) and IS (500 ng/mL) working solution was prepared by diluting its stock solution with methanol:water (50:50). Spiking solutions (200 µL) were added to drug-free human plasma (9.8 mL) as a bulk, to obtain Mozavaptan spiking concentration levels of 1.063 to 715.259 ng/mL.
The quality control pools were divided into aliquots and stored in the freezer at −60◦C until analysis. Each validation run consisted of a double quality control, system suitability sample, blank samples (a plasma sample processed without an IS), a zero sample (a plasma processed with IS), calibration curve consisting of eight non- zero samples covering the total range (1.063 to 715.259 ng/mL) and QC samples at four concentrations (n = 6, at each concentration).
Method Validation:
The method of analysis was validated as per the recommendations of ICH and FDA [1,2] for the parameters like selectivity, accuracy, precision, recovery, linearity, and stability.
Selectivity
At least six (EDTA as an anticoagulant) randomly selected control blank human plasma were be chromatographed to determine the extent to which endogenous plasma components may contribute to interference at the retention time of analyte and internal standard. Any interference is compared with the analyte response of the LLOQ.
Accuracy and Precision Within Batch
Within-batch accuracy and precision of method was performed by replicate determination of Mozavaptan concentration in human plasma. The run consisted of a calibration curve and at least six replicates of each LLOQ, low, medium and high quality control samples i.e. Precision and Accuracy Batch [P&A batch].
Between Batches
The between-batch accuracy and precision of method was performed by analysis of three precision and accuracy batch, each batch should contain at least six replicates of each LLOQ, low, medium and high quality control samples.
Accuracy and precision should be measured at minimum of four concentrations in the range of expected.
Recovery
Recovery for analyte and internal standard was performed by comparing the area of extracted samples at three different concentrations (LQC, MQC and HQC) with un-extracted standards area that represents 100% recovery.
% Recoveries at each QC concentration and of the internal standard was calculated.
Linearity
The linearity of the method was determined over calibration range of 1.014, 2.029,20.288,101.442, 326.293, 540.735, 643.733, 715.259 ng/ml The calibration standards were prepared by spiking known concentration of Mozavaptan working standard solution. A minimum of three linearity curves containing not less than six non- zero concentrations were analyzed.
Stability
Bench top stability in matrix
At least six aliquots each of the low and high quality control samples were kept at room temperature (25 + 5ºC) after spiking into plasma. After completion of 6 hours the samples were extracted and analyzed against the concentration of freshly prepared calibration curve.
Auto sampler stability
At least six aliquot of quality control samples low and high were kept remain in the auto sampler at 6ºC for 2l hours, the samples were analyzed with freshly prepared calibration standard and quality control samples. The concentration of stability samples were calculated against fresh calibration curve.
Stock solution stability
The short term stock solutions stability of analyte and internal standard were evaluated at room temperature (25+5ºC) for at least 06 hrs. Long term stability of analyte and internal standard were evaluated at refrigerated temperature (2-8ºC) for 35 and 94 days for analyte and internal standard respectively by comparing mean instrument response of the stability samples to that of comparison samples. The duration of stock solution stability should be established, if required, by periodic analysis.
Freeze and thaw stability
The freeze and thaw stability of analyte was determined after at least three freeze and thaw cycles. At least six aliquots at each of low and high quality control samples were stored at -20+5ºC and subjected to three freeze thaw cycles at an interval of 8-16 hours. After the completion of third cycle the samples were analyzed and stability of samples were compared against freshly prepared calibration curve samples.
Dilution integrity
Sample having final concentration about between three to four times of higher calibration curve standard was prepared in plasma. Then the samples were diluted 5 times and 10 times with analye free control human plasma to meet their actual concentrations in the calibration curve range. The samples were extracted along with calibration curves and results were compared with nominal concentration.
Matrix Effect
Blank matrixes obtained from three different human origins were spiked with analyte at LQC and HQC level in 3 sets. Three quality control samples of LQC and HQC from each spiked matrix along with the set of calibration standards were analysed and the % bias across the sample analysed was found to be less than + 15%.
RESULTS AND DISCUSSION
The mobile phase selected achieved a good resolution and symmetric peak shapes for the analyte and IS with a short run time. The high proportion of organic solvent eluted the Mozavaptan and Cilostazole (IS) at retention times of 1.0 and 1.9 min, respectively. A flow rate of 1.0 mL/min produced good peak shapes and permitted a run time of 2.6 min. Liquid-liquid extraction (LLE) was used for the sample preparation in this work. Diethyl ether was found to be optimal, which can produce a clean chromatogram for a blank plasma sample.
Limits of Quantitation:
The lower limit of quantitation i.e., lowest standard level with a coefficient of variation less than 20 % was 1.014 ng/ml with CV of 1.7 %, for Mozavaptan. Results are represented in table l.
The Upper limit of Quantitation for Mozavaptan was 715.25g ng/ml having CV of 2.9 %. Results are represented in table l.
Linearity:
Calibration curves were found linear over the range 1.014 to 715.259 ng/ml. For Mozavaptan co-efficient of correlation were found to be better as 0.9998. Back calculations were made from the calibration curyes to determine Mozavaptan concentrations of each calibration standard. Results are presented in table 1.
Specificity:
No significant interference was observed in 6 different lots of drug free human plasma containing EDTA as an anticoagulant, at the retention times of analyte and internal standard. Results are presented in table 2 and fig.
2,3,4.
Recovery:
Recovery of analyte
The Total recovery range of Mozavaptan was 70.9 % to 74.8%. Result are presented in table 3a.
The absolute recovery of Mozavaptan was 77.0 to 82.5 %. Result are presented in table 3b.
Recovery of Internal Standard
The Total recovery of internal standard was72.2%. Results are presented in table 4.
The absolute recovery of internal standard was 85.l %. Results are presented in table 4.
Accuracy and Precision:
Within batch
The within batch % coefficient of variation for Mozavaptan was ranged between 1.4 % to 11.9 % and accuracy was ranged between 88.8 % to 109.0 % the results are within + 15 % the results are presented in table 5.
Between-batch
The between batch % coefficients of variation for Mozavaptan was ranged between 2.8 % and 10.8 % and accuracy was ranged between 90.5 % and 97.5 %. The results are within + 15 %, the results are presented in table 6.
Stability:
Bench top stability in plasma
Percent changes (Bias) for Mozavaptan concentration for spiked samples over stability testing period of 6 hours at room temperature (25+ 5ºC) was -5.1 % to -4.6 % as compared to Nominal values. The results are within +15
%, presented in table 7.
Freeze and thaw stability
Percent change (Bias) in Mozavaptan concentration over the stability-testing period after three freeze thaw cycles at 15 + 5ºC was -6.2 % to -3.2 %. The results are within +15 %, presented in table 08.
Auto sampler stability
Percent change (Bias) in Mozavaptan concentration over the stability testing period of 21 hours at 6ºC was -3.4
% to 1.4 %. The results are within + 15 % presented in table 09.
Stock solution stability
Percent change (Bias) in Mozavaptan area response over the stability testing period of 35 day at 2-8ºC was -0.5
% and for Cilostazole solution stability for 94 days at 2-8ºC was -3.7 % (Based on Area calculation). The results are within + l0 %, presented in table 10a.
Percent change (Bias) in Mozavaptan and Cilostazole area response over the stability testing period of 06 hrs at 25+5ºC was -2.6 % and 0.6 % respectively (Based on Area calculation). The results are within + 10 % ,presented in table 10b.
Dilution Integrity:
% Accuracy and precision of.dilution integrity samples for 1/5th dilutions were 95.7% and 1.8 %o and for l/10th dilutions were 92.9 % and 2.1 %. The results are within + 15 % , presented in table 11.
Matrix effect:
Percent change (Bias) in Mozavaptan concentration in three different matrix blanks was -4.3 % to -3,3 %6.The results are within + 15 % , presented in table 12.
CONCLUSION
A simple, sensitive, and reliable LC/MS-MS method has been developed and validated for the determination of Mozavaptan in human plasma. The method is accurate, reproducible, and specific.The retention time and in-turn run time was very short, hence required less mobile phase for the method, making it more economical and rapid.
The method may be applicable for pharmacokinetic studies of Mozavaptan in human plasma.
ACKNOWLEDGEMENT
The authors thank Zydus Cadila Healthcare Ltd., Ahmedabad and Zyfine Ltd, Ahmedabad, India for providing Mozavaptan as gift sample for this work. They also thank the management of Jaipur National University for providing required facilities to carry out this research work.
REFERENCES:
[1] Food and Drug Administration. FDA. Guidance for Industry: Bioanalytical Method Validation. Rockville, MD: US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research; 2001 May.
[2] ICH Q2 (R1).Validation of Analytical Procedures: Text and Methodology. 2005.
[3] Available at http:// en.wikipedia.org/wiki/Mozavaptan. (Date-23/08/10) [4] www.drugbank.ca/drugs [Date:06/09/2010]
[5] www.pros.com/molecule/default.asp?ID=153 [Date:21/10/2010]
[6] www.bomap.info/Mozavaptan [Date:15/10/2010]
[7] Guy Decaux, Alain Soupart, Gilbert Vassart. Non-peptide arginine-vasopressin antagonists: the vaptans. The Lancet, 2008 May 10;
371(9624) 1624-1632.
[8] Punniyakoti T.V, Kenichi W, Meilei M, Palaniyandi S, Kenichi Y, Kenji S, Makoto K, Yoshifusa A.(2007) Effects of nonpeptide vasopressin V2 antagonist tolvaptan in rats with heart failure. Journal of biochemical pharmacology, 2007 July 18; 74(10) 1466-1475.
[9] Yoshitaka Y, Hidenori O, Hiroshi Y, Tomihiko C, Hisashi M, Shigeki N, Toshiyuki O, Tatsuya Y, Tetsumi H, Toyoki M, Michiaki T
& Youichi Y. Characterization of a novel aquaretic agent, Mozavaptan, as an orally effective, nonpeptide vasopressin V2 receptor antagonist. Britsh journal of pharmacology.1992 April;105(4) 787-791.
TABLES:
Table 1: Summary of back calculated concentration for each calibration curve
Back calculated calibration curve concentration Curve parameter summary Standard id STD
1
STD 2
STD 3
STD 4
STD 5
STD 6
STD 7
STD 8
r2 Slope Interce pt Nominal(ng/
mL)
1.01 4
2.02 9
20.2 88
101.4 42
362.2 93
540.7 35
643.7 33
715.2 59 Minimum 0.81
1
1.72 5
17.2 45
86.22 6
307.9 49
459.6 25
547.1 73
607.9 70 Maximum 1.21
7
2.33 3
23.3 31
116.6 58
416.6 37
621.8 45
740.2 93
822.5 48 P&A 01, RR
01 & ME 01 1.00
4
2.07 4
19.6 41
101.3 59
359.5 77
542.4 60
643.8 75
732.5 09
0.99 98
0.011 40
0.0002 57 P&A 02 &DI
01
0.98 6
2.13 9
20.3 82
102.9 37
380.1 95
538.3 97
604.4 59
693.6 21
0.99 91
0.009 54
0.0005 66 P&A 03 0.97
1
2.09 8
20.3 94
108.5 80
366.2 57
497.1 60
624.5 02
703.1 88
0.99 82
0.009 28
0.0011 10 Mean 0.98
7
2.13 7
20.1 39
104.2 92
368.6 76
526.0 06
624.2 79
709.7 73 S.D 0.01
65
0.06 20
0.43 13
3.796 4
10.51 98
25.06 35
19.70 89
20.26 30 CV% 1.7 2.9 2.1 3.6 2.9 4.8 3.2 2.9
%Nominal 97.3 105.
3
99.3 102.8 101.8 97.3 97.0 99.2
Table 2: Specificity
ANALYTE ISTD
Sr No.
BLANK Interference at RT of Analyte
LOQ area
%LOQ Interference at RT of IS
IS area % IS
1 Plasma lot-1 0.0 4370 0.0 0.0 323604 0.0
2 Plasma lot-2 0.0 5192 0.0 0.0 320241 0.0
3 Plasma lot-3 0.0 4574 0.0 0.0 336058 0.0
4 Plasma lot-4 0.0 4385 0.0 0.0 338964 0.0
5 Plasma lot-5 0.0 2832 0.0 0.0 337520 0.0
6 Plasma lot-6 0.0 3930 0.0 0.0 331557 0.0
Mean 4214 Mean 331324
SD 719.23 SD 7767.32
%CV 18.8 %CV 2.3
a) LOQ- Low Quality control b) ISTD- Internal Standard
Table 3: Recovery of Analyte from Biological Matrix
Table 3a: Total Recovery
LQC RESPONSE MQC RESPONSE HQC RESPONSE
Extracted Aqueous Extracted Aqueous Extracted Aqueous 11244 15134 1261370 1746579 1928573 2831348 11188 16332 1191646 1694978 1972660 2823183 11235 14756 1221286 1728524 1979408 2845331 11163 15039 1206782 1723371 2243381 2824738 11143 14217 1226755 1722660 2205186 2765952 11460 14651 1211194 1702788 2142058 2807224 Mean 11239 15022 1219869 1719847 2078544 2816296 S.D(+/-) 115.30 719.08 23726.84 18597.60 134739.85 27586.63
C.V (%) 1.0 4.8 1.9 1.1 6.5 1.0
% Recovery 74.8 70.9 73.8
a) LQC- Low Quality Control b) MQC- Medium Quality Control c) HQC- High Quality Control
Table 3b: Absolute Recovery
LQC RESPONSE MQC RESPONSE HQC RESPONSE
Extracted Absolute Extracted Absolute Extracted Absolute 11244 13816 1261370 1598390 1928573 2643853 11188 13238 1191646 1582187 1972660 2595320 11235 13699 1221286 1576236 1979408 2564163 11163 13555 1206782 1585210 2243381 2552854 11143 13383 1226755 1570324 2205186 2602007 11460 14047 1211194 1592872 2142058 2613480 Mean 11239 13623 1219869 1584203 2078544 2595280 S.D(+/-) 115.30 294.36 23726.84 10369.19 134739.85 33178.50
C.V (%) 1.0 2.2 1.9 0.7 6.5 1.3
% Recovery 82.5 77.0 80.1
a) LQC- Low Quality Control b) MQC- Medium Quality Control c) HQC- High Quality Control
Table 4: Recovery of Internal Standard from Biological Matrix
Sr No. Extracted Sample Un extracted Sample
Matrix Spiked(ABS)
1 399366 480489 457755
2 399534 480489 461909
3 394688 507687 462569
4 367892 534833 464963
5 381128 535336 463560
6 399137 534893 461726
7 381367 550336 458605
8 386651 545001 459274
9 390326 550970 456562
10 390065 555136 457932
11 391714 557505 456001
12 401696 551854 461299
13 384441 560247 453398
14 401783 548096 454037
15 388233 552139 457175
16 420746 56.231 461719
17 389652 555850 455987
18 398939 556300 456868
Mean 390409 540930 458963
S.D(+/-) 12193.65 22666.92 3329.58
C.V (%) 3.1 4.2 0.7
%Recovery 72.2 85.1
Table 5: Intra-batch Accuracy and Precision
CC-ID QC-ID LLOQ QC (1.063ng/mL)
LQC (3.038ng/mL)
MQC (361.664ng/mL)
HQC (623.559ng/mL)
P&A 01
& RR 01
1 1.179 5.641 311.574 610.710 2 1.385 3.121 329.106 609.642 3 1.080 3.078 328.388 625.930 4 1.222 3.032 332.208 615.712 5 0.673 3.069 325.661 640.573 6 1.028 2.953 325.333 610.658
Mean 1.159 3.051 325.378 618.871
S.D(+/-) 0.14 0.06 7.21 12.25
C.V(%) 11.9 2.1 2.2 2.0
%Nominal 109.0 100.4 90.0 99.2
Mean change 9.0 0.4 -10.0 -0.8
P&A 02
1 1.007 2.934 327.859 588.584 2 1.027 3.151 327.097 605.111 3 1.032 3.070 310.600 593.988 4 0.958 2.812 322.897 546.644 5 1.031 2.747 308.693 529.663 6 0.972 2.960 330.497 643.265
Mean 1.005 2.946 321.274 584.543
S.D(+/-) 0.03 0.15 9.35 41.05
C.V(%) 3.2 5.2 2.9 7.0
%Nominal 94.5 97.0 88.8 93.7
Mean change -5.5 -3.0 -11.2 -6.3
P&A 03
1 1.005 2.951 340.238 562.774 2 1.010 2.979 336.953 577.948 3 0.935 2.983 340.402 559.431 4 0.933 2.876 332.009 619.911 5 0.973 2.974 329.045 618.270 6 0.937 2.977 334.987 548.738
Mean 0.966 2.957 335.606 581.178
S.D(+/-) 0.04 0.04 4.53 30.82
C.V(%) 3.7 1.4 1.4 5.3
%Nominal 90.8 97.3 92.8 93.2
Mean change -9.2 -6.3 -7.2 -6.8 a) LLOQ QC- Lower Limit of Quantification Quality Control
b) LQC- Low Quality Control c) MQC- Medium Quality Control d) HQC- High Quality Control
Table 6: Inter-batch Accuracy and Precision
CC-ID QC-ID LLOQ QC (1.063ng/mL)
LQC (3.038ng/mL)
MQC (361.664ng/mL)
HQC (623.559ng/mL)
P&A 01
& RR 01
1 1.179 5.641 311.574 610.710 2 1.385 3.121 329.106 609.642 3 1.080 3.078 328.388 625.930 4 1.222 3.032 332.208 615.712 5 0.673 3.069 325.661 640.573 6 1.028 2.953 325.333 610.658
P&A 02
1 1.007 2.934 327.859 588.584 2 1.027 3.151 327.097 605.111 3 1.032 3.070 310.600 593.988 4 0.958 2.812 322.897 546.644 5 1.031 2.747 308.693 529.663 6 0.972 2.960 330.497 643.265
P&A 03
1 1.005 2.951 340.238 562.774 2 1.010 2.979 336.953 577.948 3 0.935 2.983 340.402 559.431 4 0.933 2.876 332.009 619.911 5 0.973 2.974 329.045 618.270 6 0.937 2.977 334.987 548.738 Mean 1.036 2.980 327.419 594.864
S.D(+/-) 0.11 0.10 9.25 33.56
C.V(%) 10.8 3.5 2.8 5.6
%Nominal 97.5 98.1 90.5 95.4
Mean change -2.5 -1.09 -9.5 -4.6 a) LLOQ QC- Lower Limit of Quantification Quality Control
b) LQC- Low Quality Control c) MQC- Medium Quality Control d) HQC- High Quality Control
Table 7: Bench top stability (6 hours) (at room temperature 25+5ºC)
LQC(ng/mL) HQC(ng/mL)
Nominal conc. 3.038 623.559
Bench top stability
3.071 593.596 2.893 567.884 2.912 582.861 2.853 604.436 2.809 591.482 2.760 598.932
Mean 2.883 594.865
S.D(%) 0.1075 7.4082
C.V(%) 3.7 1.2
%Nominal 94.9 95.4
%Change -5.1 -4.6 a) LQC- Low Quality Control
b) HQC- High Quality Control
Table 8: Freeze thaw Stability of Analyte in Biological Matrix (After three freeze thaw cycles) (at room temperature -20+5ºC)
LQC(ng/mL) HQC(ng/mL)
Nominal conc. 3.038 623.559
Freeze Thaw stability
2.935 600.487 2.821 596.481 2.835 603.469 2.817 609.432 2.840 611.027 1.945 600.554
Mean 2.850 603.575
S.D(%) 0.0487 5.6362
C.V(%) 1.7 0.9
%Nominal 93.8 96.8
%Change -6.2 -3.2 Samples were subjected to three freeze and thaw cycles.
a) LQC- Low Quality Control b) HQC- High Quality Control
Table 9: Auto sampler Stability of Analyte (21 Hrs.) at 6ºC.
LQC(ng/mL) HQC(ng/mL)
Nominal conc. 3.038 623.559
Auto sampler stability
3.023 633.235 2.836 645.903 2.193 628.874 2.780 647.912 3.004 612.096 3.054 626.796
Mean 2.935 632.489
S.D(%) 0.0004 13.2658
C.V(%) 3.8 2.1
%Nominal 96.6 101.4
%Change -3.4 1.4 a) LQC- Low Quality Control
b) HQC- High Quality Control
Table 10a: Stock Solution Stability at2-8ºC
ANLALYTE(35 DAYS) ISTD(94DAYS)
Stability stock Comparison stock Stability stock Comparison stock
3404049 3508009 764708 750964 3412098 3493394 763405 767084 3401342 3502281 746829 762025 3297917 3410860 755020 740378 3307549 3361597 760804 755327 3266191 3410623 773873 763517
Mean 3348191 3447794 760773 756549
S.D(%) 64703.16 61399.94 9182.10 9835.78
C.V(%) 1.9 1.8 1.2 1.3
%Stability 97.4 100.6
Mean change -2.6 0.6
Table 10b: stock solution stability for 6 hours at room temperature (25+ 5ºC)
ANLALYTE(6 hr) ISTD(6 hr)
Stability stock Comparison stock
Stability stock Comparison stock
3506718 3508009 730103 750964 3470148 3493364 718910 767084 3416086 3502281 709850 762025 3386547 3410860 727248 740378 3380468 3361597 741095 755327 3358812 3410623 743914 763517
Mean 3419797 3447794 728520 756549
S.D(%) 57403.41 61399.94 12962.64 9835.78
C.V(%) 1.7 1.8 1.8 1.3
%Stability 99.5 96.3
Mean change -0.5 -3.7
Table 11: Dilution Integrity (Five and Ten times)
Sr No.
Nominal concentration:2145.777ng/mL
Dilution fector 5 Dilution fector 10 Diluted
Concentration
Actual Concentration
Diluted Concentration
Actual Concentration
1 410.853 2054.265 199.150 1991.500
2 409.795 2048.975 191.282 1912.820
3 405.320 2026.600 202.670 2026.700
4 408.001 2040.005 200.424 2004.240
5 404.575 2022.875 201.961 2019.610
6 425.203 2126.015 200.369 2003.690
Mean 2053.123 1993.093
S.D 37.74 41.27
CV(%) 1.8 2.1
%Nominal 95.7 92.9
Mean change -4.3 -7.1
Table 12: Matrix effect
Nominal Conc.(ng/mL) LQC HQC
3.038 623.559
BLPL 01 2.805 586.903
3.013 595.628 2.904 611.747
BLPL 02 2.824 584.403
2.837 566.971 2.878 596.068
BLPL 03 2.907 602.421
3.038 641.191 2.972 639.448
Mean 2.909 602.753
S.D 0.08 24.67
%CV 2.9 4.1
%Nominal 95.7 96.7
%Bias -4.3 -3.3 a) LQC- Low Quality Control
b) HQC- High Quality Control
FIGURES:
Fig. 1: Structure of Mozavaptan
NH
CH3
N
N O
O
C H3
CH3
Fig. 2: Representative chromatogram of processed blank human plasma.
Fig. 3: Representative chromatogram of LQC.
Fig. 4: Representative chromatogram of HQC.
