Isosorbide-5-mononitrate (5-ISMN), an organic nitrate vasodilator, has been widely used worldwide to prevent angina pectoris for more than two decades. A simple and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the determination of 5-ISMN in human plasma. 13C6-5-ISMN is an internal standard, and 5-ISMN was extracted from human plasma (50
Isosorbide-5-mononitrate (5-ISMN) is an organic nitrate vasodilator and one of the long-acting metabolites of isosorbide dinitrate [
In the article, we established a simple and robust HPLC-MS/MS method for the qualification of 5-ISMN in human plasma. This analytical method had two major features which were as follows: (1) only requiring 50
The standard of 5-ISMN was obtained from the National Institutes for Food and Drug Control (purity 99.9%, China), and the standard of 13C6-5-ISMN (called internal standard, IS) was gained from TLC Pharmaceutical Standards Ltd. (purity 99.0%, Canada) (Figure
Structure of 5-ISMN (a) and 13C6-5-ISMN (b).
The chromatographic separation was carried out on LC-20A (Shimadzu, Japan) using an HPLC column ZORBAX XDB-C18 (4.6 × 50 mm, 5
The mass spectrometric detection was performed by an API 4000 mass spectrometer (AB SCIEX) under negative electrospray ionization (ESI−) source condition. The detection was operated in the multiple reaction monitoring (MRM) scan mode to analyze 5-ISMN and 13C6-5-ISMN (m/z 250.0 ⟶ 59.0 and m/z 256.1 ⟶ 58.8, respectively) (Figure
Product ion spectra of 5-ISMN (a) and IS (13C6-5-ISMN) (b).
The optimized mass spectrometric parameters.
Compound | Transition (m/z) | Declustering potential (DP) | Entrance potential (EP) | Collision energy (CE) | Collision cell exit potential (CXP) |
---|---|---|---|---|---|
5-ISMN | 250.0 ⟶ 59.0 | −25 | −10 | −20 | −3 |
13C6-5-ISMN | 256.1 ⟶ 58.8 | −25 | −10 | −20 | −3 |
Stock solutions of 5-ISMN (analyte) and its internal standard (13C6-5-ISMN) at the concentration of 0.5 mg/mL were prepared by acetonitrile-water (50 : 50,
In the European Bioanalysis Forum recommendations on the treatment of hyperlipidemia matrix [
According to the recommendation of the European Bioanalysis Forum [
The 2% hemolysis matrix was prepared as follows: freshly blank whole blood collected from 6 sources was stored at −80°C for at least one hour, then ultrasonicated at room temperature for 30 min, and vortexed for 1 min to fully rupture blood cells to obtain hemolyzed whole blood. Finally, 200
Before homogenization by vortex-mixing, frozen plasma samples were thawed thoroughly at room temperature. All the calibration standard samples, QC samples, and even clinical samples were extracted by the easy and feasible liquid-liquid extraction (LLE) method. The volume of 50
The developed method was completely validated in accordance with the US Food and Drug Administration (FDA) guidance for biological method validation [
Selectivity referred to the chromatograms of at least six individual blank human plasma samples (double blank) to assess endogenous interferences of the plasma matrix based on the corresponding retention time of analyte and IS.
Lower limit of quantification (LLOQ) was described as the lowest concentration on the calibration curve and represented the sensitivity of the analytical method. Standard samples with eight different concentrations of 5-ISMN (5.00–1000 ng/mL) constituted a calibration curve, of which linear relationship was accessed by the correlation coefficient (
Precision and accuracy on three consecutive batches completed in no less than two days were evaluated by 6 replicates at concentration level of LLOQ, LQC (low control sample), MQC (medium control sample), and HQC (high control sample) (5.00, 8.00, 80.0, and 800 ng/mL). At interbatch and intrabatch accuracy, no less than 75% of the back-calculated concentration must be in the range of 85–115% of its nominal concentration (80–120% at LLOQ).
The extraction recovery and matrix effect were evaluated by comparing the average peak areas of analyte and IS between LQC, MQC, and HQC (8.00, 80.0, and 800 ng/mL) quality control sample groups (plasma matrixes from 6 different individuals): group A (QC), extracted QC samples; group B (MQ), extracted blank matrix spiked with the corresponding concentrations of analyte and IS solutions; and group C (SQ), pure solution at equivalent concentrations as group A. Extraction recovery was calculated by QC/MQ. The absolute matrix effect was assessed by the matrix factor, which was calculated by MQ/SQ. Then, IS-normalized matrix effect was further calculated by the ratio of matrix factors between analyte and IS.
Carryover was at least evaluated by injecting a pretreated blank plasma sample after injecting the ULOQ sample of the first calibration curve in each analysis batch.
Batch capacity was confirmed by repeated injection of quality control samples to determine the maximum number of samples in an analysis batch. Taking into account some unknown sample concentrations over ULOQ, dilution integrity was investigated with a 10-fold dilution factor by using blank plasma to dilute 1600 ng/mL plasma sample of 5-ISMN to 160 ng/mL plasma sample (called dilution QC).
The stability of 5-ISMN must be evaluated at each step prior to the determination of the clinical samples to simulate as much as possible the various storage and analysis conditions of clinical samples. These stability conditions mainly included whole blood stability, autosampler stability, and repeated injection reproducibility of pretreated plasma samples, as well as short-term and long-term stability and three freeze-thaw cycles of plasma samples in different temperature. Besides, stability of stock solution and working solutions at room temperature or under freezing conditions (−80oC) were also carried out.
Considering the actual situation of clinical operation, freshly collected clinical samples may undergo hemolysis during storage or pretreatment, so it was necessary to assess the precision and accuracy of the analyte in the hemolysis matrix. Because of the bioequivalence evaluation of 5-ISMN containing fed trial, it was necessary to investigate the determination of the analyte influenced by the hyperlipidemia matrix.
Data acquisition and data management were run through Analyst (version 1.5.1, AB SCIEX, USA) and Watson LIMS software (version 7.3, Thermo Scientific), respectively. The linear regression of calibration curves was weighted by 1/
The bioequivalence study of isosorbide 5-mononitrate (5-ISMN) tablets in healthy Chinese people was under fasting and fed conditions, respectively. This bioequivalence study was designed as an open, single-center, single-dose, two-cycle, two-sequence, randomized, crossover trial. The study protocol was approved by the Ethics Committee of People’s Hospital of Wuxi (Jiangsu, China), and all the subjects signed informed consent before the study. A total of 56 healthy subjects participated in the study and received a single dose of 20 mg 5-ISMN tablets. Blood samples were obtained after administrating the 20 mg 5-ISMN tablet at specific time points within 36 h. Each subject had 19 blood collection points for the fasting condition and 18 blood collection points for the fed condition per cycle. To obtain plasma samples, the blood samples were drawn into tubes including anticoagulant and then centrifuged at 3000 rpm at 4°C within 10 min to take the supernatant. The above clinical plasma samples were frozen under −80°C condition before analysis.
According to the bioanalytical guidelines from the FDA [
In the article, a rapid, specific, and robust HPLC-MS/MS method was developed for the determination of 5-ISMN in human plasma. It was known that stable isotope-labeled compounds had similar structure and physicochemical properties to target analytes to reduce matrix effects; therefore, 13C6-5-ISMN was used for the internal standard to more accurately quantify the concentration of 5-ISMN. During the exploration of mass spectrometry conditions, an API 4000 mass spectrometer (AB SCIEX, USA) was used to detect the target analytes with the positive and negative ionization mode (ESI+ or ESI−). After the MS parameters reached the optimal conditions, it was turned out to be weak or inconsistent response at m/z 192.1 [M + H] or 190.1 [M−H], using 5-ISMN solution in acetonitrile or acetonitrile/water (
Chromatographic separation was constructed on LC-20A (Shimadzu, Japan) equipped with an API 4000 mass spectrometer. During method development, ZORBAX XDB-C18 was a commonly HPLC column for the widest range of compound species. After changing the different ratios of mobile phases and different flow rates, it turned out that 5-ISMN and IS had excellent peak shape and retention time when ZORBAX XDB-C18 (4.6 × 50 mm, 5
Figure
Representative MRM chromatogram of 5-ISMN and IS (13C6-5-ISMN, 1000 ng/mL). (a) Blank plasma, (b) blank plasma spiked with IS, (c) LLOQ plasma sample, and (d) unknown clinical plasma sample.
Calibration curves were established within the concentration ranges of 5.00–1000.00 ng/mL for 5-ISMN in plasma samples. Correlation coefficients (r) over 0.99 with RSD 0.2% proved that all the calibration curves had an excellent linear relationship (Supplementary Figure
Intraday and interday precision and accuracy of the analytical method were determined by testing different concentrations of independent QC samples in plasma. As the specific results are shown in Table
Intraday and interday accuracy and precision of quality control samples of 5-ISMN.
Nominal con. (ng/ml) | Intraday ( | Interday ( | ||||
---|---|---|---|---|---|---|
Measured con. (mean ± SD, ng/mL) | Precision (RSD %) | Accuracy (%) | Measured con. (mean ± SD, ng/mL) | Precision (RSD %) | Accuracy (%)a | |
5 | 4.64 ± 0.23 | 4.8 | -7.2 | 4.56 ± 0.30 | 6.6 | −8.8 |
8 | 8.22 ± 0.44 | 5.3 | 2.8 | 8.13 ± 0.44 | 5.4 | 1.6 |
80 | 85.7 ± 4.01 | 4.7 | 7.1 | 82.2 ± 3.31 | 4.0 | 2.8 |
800 | 824 ± 31.2 | 3.8 | 3.0 | 817 ± 19.2 | 2.4 | 2.1 |
Concentration is in 3 significant figures; SD, standard deviation; RSD%, percent relative standard deviation (in one decimal place). aExpressed as ((mean measured concentration−nominal concentration)/(nominal concentration)) × 100 (in one decimal place).
After a pretreatment method of liquid-liquid extraction using ethyl acetate, the mean extraction recovery of 5-ISMN at three QC levels was 87.0% with 7.0% RSD and that of IS (13C6-5-ISMN) was 80.9% with 9.7% RSD. The matrix effect was calculated by the peak area ratio of the analyte spiked with extracted blank matrix, to pure solution of equivalent concentrations. The matrix effect of 5-ISMN of LQC, MQC, and HQC samples ranged from 87.0% to 95.5% and the IS-normalized matrix effect of 5-ISMN was 98.6% to 107.8% under the present analytical condition. Table
Extraction recovery and matrix effect of 5-ISMN in plasma (
Compound | Nominal con. (ng/mL) | Recovery (%) | Mean recovery% (RSD %) | Matrix effect (%) | IS-normalized matrix effect | Mean matrix effect% (RSD %) | |
---|---|---|---|---|---|---|---|
5-ISMN | IS | ||||||
5-ISMN | 8 | 82.6 | 87.0 (7.0) | 95.5 | 88.6 | 107.8 | 102.1 (0.85) |
80 | 84.3 | 87.0 | 88.3 | 98.6 | |||
13C6-5-ISMN | 800 | 93.8 | 93.4 | 94.3 | 99.8 | ||
1000 | 80.9 | NA | NA | NA | NA | 102.1 (0.67) |
Concentration is in 3 significant figures; RSD%, percent relative standard deviation (in one decimal place). NA, not available.
For carryover evaluation, the peak area of 5-ISMN and 13C6-5-ISMN (IS) in the blank plasma sample following the ULOQ (upper limit of quantification) sample did not exceed 6.2% and 5.0% of the peak area of LLOQ, respectively, indicating no significant carryover.
After repeated injections of LQC, MQC, and HQC samples of an eligible analytical batch for 9 times, the batch capacity of one analysis batch capable of analyzing the maximum number of samples was determined to be 162. This facilitated the efficient and rapid determination of the concentration of clinical samples.
Similarly, the result of the dilution integrity test presented that QC samples could be diluted with dilution factor 10 (mean values for precision did not exceed ±5.8% with an accuracy (RE) of −6.8%).
Table
Stability assessments of 5-ISMN in plasma (
Matrix | Storage conditions | Nominal con. (ng/mL) | Measured con. (mean ± SD, ng/mL) | Precision (RSD %) | Accuracy (%)a |
---|---|---|---|---|---|
Blood | Room temperature for 2 h | 8 | 7.76 ± 0.18 | 2.3 | −2.5 |
200 | 212 ± 5.18 | 2.4 | 6.0 | ||
Plasma | Three circles of freeze/thaw | 8 | 8.60 ± 0.35 | 4.1 | 7.5 |
800 | 833.2 ± 8.82 | 1.1 | 4.1 | ||
In autosampler for 72 h after extraction | 8 | 8.62 ± 0.43 | 5.1 | 7.8 | |
800 | 823.2 ± 24.53 | 3.0 | 2.9 | ||
Room temperature for 8 h | 8 | 7.90 ± 0.35 | 4.5 | −1.3 | |
800 | 823.5 ± 7.23 | 0.9 | 2.9 | ||
Long-term stability (−30°C for 63 days) | 8 | 7.49 ± 0.53 | 7.1 | −6.4 | |
800 | 820.5 ± 26.85 | 3.3 | 2.6 | ||
Long-term stability (−80°C for 91 days) | 8 | 8.31 ± 0.19 | 2.2 | 4.0 | |
800 | 755.67 ± 9.93 | 1.3 | −5.5 | ||
Repeat injection for 64 h after extraction | 8 | 7.71 ± 0.22 | 2.9 | −3.6 | |
800 | 821.7 ± 25.12 | 3.1 | 2.7 |
Concentration is in 3 significant figures; SD, standard deviation; RSD%, percent relative standard deviation (in one decimal place). aExpressed as ((mean measured concentration−nominal concentration)/(nominal concentration)) × 100 (in one decimal place).
As shown in Table
Precision and accuracy of 5-ISMN in hyperlipidemia and hemolysis matrices (
Matrix | Nominal con. (ng/mL) | Measured con. (mean ± SD, ng/mL) | Precision (RSD %) | Accuracy (%)a |
---|---|---|---|---|
300 mg/dL hyperlipidemic matrix | 8 | 7.79 ± 0.37 | 4.7 | −2.6 |
80 | 82.5 ± 3.89 | 4.7 | 3.1 | |
800 | 831 ± 23.18 | 2.8 | 3.8 | |
2% hemolysis matrix | 8 | 7.91 ± 0.46 | 5.8 | −1.1 |
80 | 80.8 ± 2.74 | 3.4 | 1.0 | |
800 | 852 ± 14.15 | 1.7 | 6.5 |
Concentration is in 3 significant figures; SD, standard deviation; RSD%, percent relative standard deviation. aExpressed as ((mean measured concentration−nominal concentration)/(nominal concentration)) × 100 (in one decimal place).
The completely validated method was efficiently applied to the bioequivalence study of isosorbide 5-mononitrate (5-ISMN) tablets in healthy Chinese people under fasting and fed conditions, respectively. About 2,000 clinical samples from a total of 56 healthy subjects were efficiently analyzed in a week. Representative chromatogram of 5-ISMN in a clinical sample at 6 h after a single dose of 20 mg 5-ISMN is shown in Figure
The mean concentration-time curves of reference and test drugs in Chinese healthy subjects after a single dose of the 20 mg 5-ISMN tablet in fasting condition (
105 clinical samples for 5-ISMN under the fasting condition and 108 clinical samples for 5-ISMN under the fed condition were reanalyzed, respectively. As shown in Figure
Percentage difference of reanalysis result versus mean result for 5-ISMN of clinical plasma samples in fasting condition (
A sensitive and rapid LC-MS/MS method was developed and validated for the determination of 5-ISMN in human plasma. This method was practical and quickly applied to the bioequivalence study of oral 20 mg 5-ISMN tablets in Chinese volunteers.
All data used to support the findings of this study will be made available from the corresponding author upon reasonable request.
Yinping Zhou and Aijing Liu are the co-first authors. Qing He is the co-corresponding author.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
All the authors are listed, and they have contributed substantially to the manuscript. Yinping Zhou and Aijing Liu contributed equally to this study.
This study was supported by the Chinese National Major Project for New Drug Innovation (2019ZX09734001) and Livzon Pharmaceutical Group Inc. (Zhuhai, China).
Supplementary Figure 1 shows the representative calibration standards within the concentration ranges of 5.00–1000.00 ng/mL for 5-ISMN in plasma samples, and Supplementary Table 1 summarizes the correlation coefficients (