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Quantification of Isosorbide 5-mononitrate in Human Plasma by Solid Phase Extraction and LC/MS/MSW. Zuo, J. Qiao, Q. Yang
MicroConstants China Inc., Beijing, China 102206
Introduction
Isosorbide 5-mononitrate (5-ISMN) is an organic nitrate widely used for its vasodilating properties in the treatment of angina pectoris. 5-ISMN is also one of the metabolized forms of isosorbide dinitrate which is pharmacologically active. A rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/MS/MS) method for 5-ISMN in human plasma is described.
5-ISMN Concentration (ng/mL) in Human Plasma Calibration Curve Data and Statistics
Analytical Run
Standard Concentration (ng/mL)5.00 10.0 20.0 50.0 100 200 500 1000 5000
AR01 4.32 5.47 11.1 20.3 48.7 96.7 198 497 980 5120 5050AR02 4.96 5.05 10.3 20.4 49.4 96.6 195 488 1000 5100 5100AR03 5.70 4.95 9.82 19.2 47.5 97.9 190 497 1000 5190 5150AR04 5.67 5.04 10.0 19.4 46.9 95.0 191 474 989 5410 5210
Mean 5.15 10.3 19.8 48.1 96.6 194 489 992 5170%CV 8.88 5.49 3.09 2.35 1.23 1.91 2.22 0.975 2.15%DEV 3.00 3.00 -1.00 -3.80 -3.40 -3.00 -2.20 -0.800 3.40
Analytical Method Summary
Instrumentation Agilent 1100 System coupled to Quattro Ultima tandem quadrupole mass spectrometer
Column Waters Atlantis dC18 150×2.1 mm, 5 μm, analytical column
Analyte Isosorbide 5-Mononitrate (5-ISMN)
Internal Standard Isosorbide-13C6 5-Mononitrate
Ionization Mode Electrospray Negative, Multiple Reaction Monitoring (MRM)
Mass Transitions 5-ISMN: 249.90>59.04 (as acetate adduct)I.S.: 255.97>59.11 (as acetate adduct)
HPLC Mobile Phase 1.00 mM Ammonium Acetate, 0.02% Acetic Acid in Water:Methanol (65:35, v/v)
Sample Extraction Volume 500 μL
Calibration Curve Range 5-ISMN: 5.00 to 5,000 ng/mL
Anticoagulant Sodium Citrate
Retention Times 5-ISMN, I.S.: 3.00min
SPE Cartridge Oasis HLB (30 mg/1mL) extraction cartridge, Waters
Sample Extraction Method Cartridges were conditioned with 1 mL of methanol, then equilibrated with 1 mL of water (x2). Samples were loaded, pulled through the cartridge, washed with 1 mL of water (x2), dried briefly, then eluted with 0.2 mL of methanol. The eluent was diluted with 0.05 mL of water, mixed, and an aliquot was injected onto the LC system.
Representative Calibration Curve (5.00 to 5,000 ng/mL) for 5-ISMN
in Human Plasma
Stability5-ISMN Theoretical
Concentration (ng/mL)
Reinjection Integrity 118 Hours at 5oC
Five Freeze/Thaw Cycles
25 Hours Thawed At Room Temperature
15.0 1.33 to 6.00 2.67 2.004,000 4.25 to 8.50 9.75 7.50
Reinjection integrity of prepared samples was tested by re-injection of a calibration curve and QC samples in sextuplicate. The stability of the freeze/thaw, thawed stability samples was tested by ex-tracting and analyzing QC samples in triplicate.
HPLC Carry-Over Evaluation
Analytical Run
Lowest LLOQ Peak Height
Replicate
Peak Height Carry-Over Percentage of LLOQ
Peak Height*ULOQ
Carry-Over Blank
MB02r 8,367 1 12,478,853 0 0.0%2 12,284,856 0 0.0%3 11,520,672 2,488 29.7%4 12,193,975 0 0.0%5 11,503,737 0 0.0%6 11,613,800 0 0.0%
Mean - - 11,900,000 415 4.96%
* Following injection of a unique ULOQ sample (5,000 ng/mL)
Carry over potential was evaluated by injecting six plasma blank samples immediately following a unique ULOQ sample, the mean response of the carry-over blanks was less than 20.0% of the LLOQ peak height indicating acceptable prevention of carry-over between injections.
FIP Pharmaceutical Sciences 2010 World Congress in Association with the AAPS Annual Meeting & Exposition | Nov. 14-18, 2010 | New Orleans, LA | Poster #T2331
Structure of 5-ISMN
Representative Chromatograms for 5-ISMN in Human Plasma
LLOQ (5.00 ng/mL) Blank Plasma ULOQ (5,000 ng/mL)
Accuracy and Precision
Interday Theoretical Concentration (ng/mL)
Intraday Theoretical Concentration (ng/mL)
15.0 150 4,000 15.0 150 4,000
Mean 16.0 154 4,240 Mean 15.6 152 4,300%CV 4.51 3.30 2.94 %CV 3.02 2.24 1.51%DEV 6.67 2.67 6.00 %DEV 4.00 1.33 7.50
Interday accuracy and precision were determined by analyzing six replicate QC samples in human plasma at three concentrations over the course of four separate analyses. Intraday accuracy and precision were determined by analyzing six replicate QC samples in human plas-ma at three concentrations in a single run.
Matrix EffectTheoretical
Concentration (ng/mL)
Plasma Lot Number
ReplicateReported
Concentration (ng/mL)
%DEV Lot Acceptance
5.00 SC10001 1 4.80 -4.00 Pass2 5.13 2.603 5.06 1.20
SC10002 1 5.74 14.8 Pass2 5.54 10.83 5.23 4.60
SC10003 1 4.69 -6.20 Pass2 5.33 6.603 5.23 4.60
SC10004 1 5.59 11.8 Pass2 4.62 -7.603 5.91 18.2
SC10005 1 5.72 14.4 Pass2 5.46 9.203 5.52 10.4
SC10006 1 5.45 9.00 Pass2 5.84 16.83 5.42 8.40
Overall Acceptance 100%
The impact of matrix effect on the analysis of 5-ISMN was determined by calculating the %DEV of three replicates for each lot spiked at the LLOQ standard concentration level.
ConclusionsA robust and simple method for the determination of 5-ISMN in human plasma has been developed. The method will be used for a bioequivalence study.