Synchronized determination of sacubitril and valsartan with some co-administered drugs in human plasma via UPLC–MS/ MS method using solid-phase extraction
Bahia Abbas Moussa|Hanaa M. A. Hashem|Marianne Alphonse Mahrouse| Sally Tarek Mahmoud
Abstract
An accurate and sensitive UPLC–MS/MS method was developed and validated for the simultaneous estimation of the newly developed combination of sacubitril and valsartan and the co-administered drugs nebivolol, chlorthalidone and esomeprazole in human plasma. Solid-phase extraction was conducted for the purification and extraction of the drugs from human plasma. Chromatographic separation was carried out on an Agilent SB-C18 (1.8 μm, 2.1 50 mm) column using losartan as internal standard. Isocratic elution was applied using acetonitrile–0.1% formic acid in water (85: 15, v/v) as mobile phase. Detection was carried out using a triple-quadrupole tandem mass spectrometer using multiple reaction monitoring, at positive mode at m/z 412.23 ! 266.19 for sacubitril, m/z 436.29 ! 235.19 for valsartan, m/z 405.8 ! 150.98 for nebivolol, m/z 346.09 ! 198 for esomeprazole and a selected combination of two fragments m/z 423.19 ! 207.14 and 423.19 ! 192.2 for losartan (internal standard), and in negative ionization mode at m/z 337.02 ! 190.12 for chlorthalidone. The method was linear over the concentration ranges 30–2,000 ng/ml for sacubitril, 70–2,000 ng/ml for valsartan, esomeprazole and chlorthalidone and 70–5,000 pg/ml for nebivolol. The developed method is sensitive and selective and could be applied for dose adjustment, bioavailability and drug–drug interaction studies.
KEYWORDS
chlorthalidone, esomeprazole, nebivolol, sacubitril, solid-phase extraction, valsartan
1 | INTRODUCTION
The combination of sacubitril (SAC, Figure 1a) and valsartan (VAL, Figure 1b) is a first-in-class neprilysin angiotensin receptor inhibitor for the treatment of patients who suffer from chronic heart failure as a result of hypertension with a lower-than-normal ejection fraction (Vardeny et al., 2014). Its ability to augment the endogenous natriuretic peptide system and inhibit the renin angiotensin– aldosterone axis provides a distinctive mechanism of action in cardiovascular disease. According to the guidelines for the treatment of patients with hypertension and heart failure, the concomitant administration of two or more drugs depending on patient needs is recommended (McMurray et al., 2012). This treatment strategy reports that the use of angiotensin receptor blocker and a betablocker is essential in modifying the course of systolic heart failure and should be considered in every patient. They are commonly used in conjunction with a diuretic, which plays a role in relieving the symptoms and signs of congestion. Nebivolol (NEB, Figure 1c) is a highly selective beta1-adrenergic blocker that leads to vasodilation and decreased peripheral vascular resistance. Studies suggest that long-term therapy with NEB improves left ventricular function and exercise capacity in patients with stable heart failure (Veverka & Salinas, 2007). Chlorthalidone (CLT, Figure 1d) is a thiazide like diuretic that is recommended to relieve edema in heart failure patients. Moreover, low-dose aspirin therapy plays an important role in both the primary and secondary prevention of cardiovascular events, as established by the American Heart Association. The base of the use of aspirin for primary prevention is the evaluation of the patient’s risk of cardiovascular events compared with their risk of adverse events, such as bleeding. Therefore, the use of a proton pump inhibitor such as esomeprazole (ESO, Figure 1e) with lowdose aspirin therapy in patients at high risk of developing gastric ulcers for the prevention of cardiovascular disease significantly reduced their risk of ulcer development (Mehra et al., 2013). From the aforementioned guidelines, SAC/VAL combination frequently can be co-administered with NEB, CLT and ESO for the treatment of patients with heart failure.
A literature review revealed that the SAC/VAL combination has been estimated by spectrophotometric (Eissa & Abou Al Alamein, 2018) and spectrofluorimetric methods (Ragab et al., 2017) in their combined tablets. HPLC methods have also been reported for the determination of the binary mixture in bulk powder, pharmaceutical formulations and biological fluids using different detectors such as diode array (Ragab et al., 2018), UV (Naazeen & Sridevi, 2017; Patel et al., 2016; Zhou et al., 2018) and tandem mass (Ayalasomayajula et al., 2015, 2016, 2017; Chunduri & Dannana, 2016; Gan et al., 2016; Gu et al., 2010; Han et al., 2017; Hsiao et al., 2015). However, the aforementioned HPLC–MS/MS methods focus on pharmacokinetic studies (Chunduri & Dannana, 2016; Gu et al., 2010) in rat plasma and the assessment of the drug–drug interaction potential of SAC/VAL with other drugs such as digoxin or warfarin (Ayalasomayajula et al., 2015), hydrochlorothiazide, amlodipine or carvedilol (Hsiao et al., 2015), omeprazole, metformin (Gan et al., 2016) and atorvastatin (Ayalasomayajula et al., 2017). No method has been reported for the simultaneous determination of SAC/VAL in human plasma in the presence of the cited co-administered drugs recommended by the guidelines for treatment of heart failure. Therefore, an appropriate and feasible analytical method to determine the concentrations of the five drugs in human plasma is needed, which is also of importance for monitoring and controlling the dose in heart failure patients.
In the present work, a novel validated UPLC–MS/MS method coupled with solid-phase extraction (SPE) was established and validated for the simultaneous estimation of five drugs (SAC, VAL, NEB, CLT and ESO) in human plasma. a Superior sample preparation method using an advanced strategy for the extraction of drugs of variable polarities was followed employing Oasis® HLB cartridges. This simple strategy allowed effective plasma extraction and purification. The developed method could be applied for dose adjustment, bioavailability and drug–drug interaction studies.
2 | EXPERIMENTAL
2.1 | Materials and chemicals
Sacubitril and VAL were purchased from Shanxi Jinjin Chemical Co. Ltd. Their purities were 99.10 ± 0.87 and 99.50 ± 0.73, respectively. Losartan (internal standard, IS), NEB, CLT and ESO were kindly supplied by the National Organization of Drug Control and Research, Giza, Egypt and were certified to contain 99.29, 101.16, 99.67 and 100.21%, respectively. Human plasma was purchased from the Holding Company for Biological Products and Vaccines. Acetonitrile (HPLC grade) formic acid, o-phosphoric acid and methanol (Sigma-Aldrich Co., Germany) were used. Bi-distilled water was produced in-house (Aquatron Water Still, A4000D, UK).
2.2 | Instrumentation
An LC–MS/MS system (Waters TQD system), equipped with vacuum degasser, gradient quaternary-pump, auto-sampler, column oven and triple-quad tandem mass detector, was employed. Data acquisition and processing were performed using Masslynx Workstation software (4.1 SCN 805). An Eppendorf concentrator 5301, (USA), a Vortex Stuart “Bio cote” (UK) and a Centrifuge (OHAUS, Frontier 5,706, Germany) were used. The SPE was performed on a Waters SPE manifold, using Waters Oasis® HLB extraction cartridges (1 ml containing 30 mg of the sorbent, USA). Samples were evaporated under nitrogen using a FlexiVap Work Station (model 109 A YH-1, Glas-Col Tools for Scientists, USA). A sonicator (Power Sonic 405, Human Lab, Gwangju-si, Gyeonggi, Korea) was used.
2.3 | Chromatographic and mass spectrometric conditions
Chromatographic elution was performed using a mobile phase consisting of acetonitrile–0.1% formic acid in water (85: 15, v/v) pumped through an Agilent SB-C18 column (1.8 μm, 2.1 50 mm), at a flow rate of 0.2 ml/min. Under these conditions, the total run time was 3 min and the retention times for the IS, Val, SAC, ESO, CLT and NEB were approximately 0.69, 0.71, 0.76, 0.71, 0.65 and 1.55 min, respectively (Figure 2b).
The Waters TQD LC–MS/MS system was connected to the chromatographic system using an electrospray ionization interface and was operated in positive ion mode for the detection of SAC, VAL, NEB, ESO and IS and in negative mode for CLT. The desolvation gas flow was set at 600 L/h and desolvation temperature was 400C. Source temperature was set at 150C. Multiple reaction monitoring transitions were measured in positive ion mode at m/z 412.23 ! 266.19 for SAC, m/z 436.29 ! 235.19 for VAL, m/z 405.8 ! 150.98 for NEB, m/z 346.09 ! 198 for ESO and m/z 423.19 ! 207.14 and 423.19 ! 192.2 for losartan (IS) and at negative ion mode at m/z 337.02 ! 190.12 for CLT, with a 0.020 s dwell time for all ions. The cone voltage was set at +25 V for SAC, +20 V for VAL, NEB, ESO and losartan (IS) and +35 V for CLT. The collision energy was set at 30 V for SAC, 25 V for VAL, 55 V for NEB, 15 V for ESO, 25 V for CLT and 35 V for losartan (IS). Quantitation of the drugs in human plasma was based on the peak area ratio of the cited drug vs. that of the IS. Data acquisition and processing were performed using Masslynx Workstation software (4.1 SCN 805).
2.4 | Preparation of the standard stock solutions and working solutions
Two stock solutions of SAC (200 μg/ml) in a mixture of acetonitrile and water (95: 5, v/v), and of VAL (200 μg/ml), CLT (200 μg/ml), ESO (200 μg/ml) and NEB (500 ng/ml) in acetonitrile were prepared in a series of 100 ml volumetric flasks, one set for the preparation of calibration standards and another for the preparation of quality control (QC) samples. Losartan (IS) stock solution (100 μg/ml) was prepared in acetonitrile and further diluted with the same solvent in order to obtain losartan working solution (100 ng/ml).
2.5 | Preparation of the calibration standards and quality control samples
Aliquots (50 μl) of the prepared serial dilutions (600–40,000 ng/ml) from SAC stock solution, (1.4–40 μg/ml) from VAL, CLT and ESO stock solutions and (1.4–100 ng/ml) from NEB stock solution were transferred into five series of Wassermann tubes containing 450 μl of drug free human plasma. Aliquots (20) μl of IS solution and (480 μl) of 4% o-phosphoric acid were added to each tube in order to prepare six calibration standards in the ranges 30–2,000 ng/ml for SAC, 70–2,000 ng/ml for VAL, CLT and ESO and 70–5,000 pg/ml for NEB. The quality control (QC) samples were prepared similarly to the calibration standard at three concentration levels—low (QCL), medium (QCM) and high (QCH): 90, 800 and 1,600 ng/ml for SAC; 210, 800 and 1,600 ng/ml for VAL, CLT and ESO; and 210, 2,500 and 4,000 pg/ml for NEB. All samples were vortex mixed to ensure complete mixing. During each run, three replicates of QC samples were used along with the calibration standards to verify the reproducibility, repeatability and integrity of the method.
2.6 | Solid-phase extraction of plasma samples
After spiking the plasma with the drugs and IS, aliquots (480 μl) of 4% o-phosphoric acid were added and the samples were vortex mixed for 2 min. Solid-phase cartridges were conditioned using 1 ml methanol followed by equilibration using 1 ml water. Then, the acidified samples were slowly loaded onto the solid-phase cartridges. The cartridges were washed with 1 ml of 5% methanol in water. The cited drugs and IS were eluted using 1 ml methanol. The collected samples were then evaporated to dryness in a concentrator under a stream of nitrogen at 45C. The obtained residue was reconstituted in 100 μl of mobile phase, vortex mixed and transferred into a glass vial for LC–MS/MS analysis.
2.7 | Method validation
The bioanalytical method was validated in accordance with the guidelines of the European Medicines Agency (2012), involving linearity and determination of the lower limit of quantitation (LLOQ), selectivity, precision, accuracy, matrix effect, dilution integrity and stability.
2.7.1 | Linearity and lower limit of quantitation
For construction of the calibration curve of each drug, a series of calibration standards (30–2,000 ng/ml for SAC, 70–2,000 ng/ml for VAL, CLT and ESO and 70–5,000 pg/ml for NEB) were prepared as described in Section 2.5. Five linearity curves containing six concentrations were constructed by plotting the peak area ratio of the cited drugs to the IS vs. the corresponding concentration. The lowest concentration in the calibration curve could be defined as the LLOQ if the drug response was five times that of drug-free (blank) plasma, the variation in the precision did not exceed 20% and the accuracy was within 80–120%.
2.7.2 | Selectivity
Six randomly selected drug-free human plasma samples were processed by SPE as described in Section 2.6 and were analyzed using the chromatographic conditions stated in Section 2.3. The chromatograms produced were compared with those obtained by analyzing each drug at LLOQ level to determine the extent to which endogenous plasma components may interfere at the retention times of the drugs and the IS.
2.7.3 | Intra- and interday precision and accuracy
Intraday precision and accuracy were evaluated by three replicate analyses of SAC, VAL, ESO, CLT and NEB at LLOQ, QCL, QCM and QCH concentrations in human plasma on the same day. The interday precision and accuracy were assessed by analysis of three replicates of LLOQ, QCL, QCM and QCH samples for SAC, VAL, ESO, CLT and NEB on three consecutive days. The precision and accuracy of the method were determined by calculating the percentage coefficient of variation (CV) and percentage recovery for the concentrations obtained for different determinations (European Medicines Agency, 2012).
2.7.4 | Matrix effect
The matrix effect of the proposed method was assessed by calculation of the matrix factor (MF) of each drug and IS and also by calculation of the IS-normalized MF. The MF was calculated for each human plasma matrix using six drug-free human plasma samples spiked with the drug (after the extraction process) followed by direct comparison of the mean peak areas of QC samples of low and high concentrations with the mean peak areas of the plain standard solutions of equivalent concentration. The IS-normalized MF was calculated by dividing the MF of each drug by the MF of the IS.
2.7.5 | Dilution integrity test
Dilution of samples with blank human plasma should not affect the accuracy and precision of the method. Therefore, two factors were used to demonstrate the effect of dilution on the proposed method by spiking the human plasma matrix with each drug at concentrations above the upper limit of quantification (ULOQ; 4,000 ng/ml for SAC, VAL, ESO and CLT and 10,000 pg/ml for NEB) and then diluting each sample 2- and 4-fold with blank human plasma, each in six replicates. Then the samples were processed and precision and accuracy were calculated.
2.7.6 | Stability
Short-term stability was evaluated by keeping three aliquots each of the unprocessed LQC and HQC samples at ambient temperature for 6 h. After 6 h, the samples were processed, analyzed and compared with the nominal concentrations of freshly prepared QC samples. For long-term stability, three aliquots each of QCL and QCH samples were stored in deep freezer at 80 ± 2C for 15 days. After 15 days, the samples were processed and analyzed with freshly prepared QC samples immediately analyzed after preparation. The obtained concentrations were compared with the nominal concentrations of freshly prepared QC samples to determine the long-term stability of SAC, VAL, ESO, CLT and NEB in human plasma. Autosampler stability was determined by analyzing three aliquots each of QCL and QCH samples that were processed and reconstituted with mobile phase before storing for 24 h in the autosampler. After 24 h, the samples were reanalyzed and concentrations were compared with the freshly prepared control samples for the analytes. The area of the IS obtained from the analysis of samples after 24 h was compared with the area of the IS obtained from freshly prepared control samples. The effect of freeze–thaw cycles on stability of plasma samples after three cycles was also determined. Three aliquots each of LQC and HQC samples were stored at 80 ± 2C and subjected to three freeze–thaw cycles. At each cycle, samples were frozen for at least 12 h before they were thawed unassisted. After the completion of the third cycle, the samples were processed and analyzed and the results were compared with nominal values of freshly prepared QC samples.
3 | RESULTS AND DISCUSSION
The present investigation enabled the simultaneous determination of the new combination SAC/VAL and the co-administered drugs in human plasma by UPLC–M/MS. SPE was employed to ensure effective plasma extraction and avoid matrix effect. Based on the Cmax value (Andersson et al., 2001; Ayalasomayajula et al., 2018; Kamali et al., 1997) of the cited drugs, suitable linearity ranges of the five studied drugs were selected. This allowed feasibility of method application to dose adjustment in heart failure patients, in addition to bioavailability and drug–drug interaction studies.
3.1 | Method development
3.1.1 | Optimization of chromatographic and mass spectrometric conditions
In view of importance of including an IS in LC–MS/MS analysis, different ISs such as candesartan and losartan were tested. Losartan was the IS of choice as it provided optimum response with no interference with the studied drug.
Preliminary tests were performed to select optimum conditions for the determination of SAC, VAL, ESO, CLT and NEB in human plasma. Several chromatographic conditions were attempted using various mobile phase compositions. Organic modifiers such as methanol or acetonitrile were tried in different proportions with water, 0.1% formic acid in water and ammonium acetate buffer, in an isocratic mode. Acetonitrile allowed better elution of the drugs with sensitive response while formic acid (0.1%) enabled a higher detection response through assisting the ionization of the cited drug molecules.
Therefore, a mobile phase consisting of acetonitrile–0.1% formic acid in water (85: 15, v/v), at a flow rate 0.2 ml/min, using isocratic conditions was the selected as the mobile phase of choice since it permitted good elution of the drugs and IS.
On the other hand, mass spectrometric conditions were optimized in order to obtain a sensitive and stable signal response of mass transition for the drugs and IS. All of the drugs were ionized using an electrospray ionization source prior to detection in multiple reaction monitoring mode which was conducted at the following transitions: in positive ionization mode at m/z 412.23 ! 266.19 for SAC, m/z 436.29 ! 235.19 for VAL, m/z 346.09 ! 198 for ESO and m/z 405.8 ! 150.98 for NEB and in negative ionization mode at m/z 337.02 ! 190.12 for CLT (Figure 3). Regarding the choice of IS transitions, a challenge was encountered owing to the large variation between peak area responses of SAC, VAL and ESO on one side and NEB and CLT on the other side. By studying the fragmentation patterns of the IS, two characteristic daughter ions at 207.14 and 192.2 were observed, where the first one had a higher peak area response than the second one. Therefore, two multiple reaction monitoring transitions in positive ionization mode for IS were selected; m/z for NEB and CLT. The selected combination of two fragments of the IS avoided the use of two different ISs with different peak area responses to match those of the drugs.
3.1.2 | Optimization of plasma sample extraction protocol
The sample preparation process is a critical step in bioanalysis to remove any interference from a matrix, such as plasma in our study, which contains a large amount of phospholipids (Vaghela et al., 2016). Liquid–liquid extraction is time consuming and not suitable for polar drugs, while protein precipitation leads to significant ion suppression in mass spectrometry. On the other hand, SPE overcomes many of the problems of other extraction methods. It depends on using a chromatographic packing material, usually in a cartridge-type device, to separate the different components of a sample according to physicochemical properties of the drugs and the packing material (Simpson, 2000). Solid-phase extraction was adopted in the present method for sample preparation. An Oasis® HLB (hydrophilic–lipophilic balanced copolymer) cartridge was the cartridge of choice in this study. The sorbent composition made it universal for acidic, basic and neutral compounds. Therefore, it was suitable for the separation of the cited drugs as they vary in acidic and basic characteristics. The extraction protocol was adjusted. The plasma samples were loaded over the cartridges (preconditioned with 1 ml methanol) followed by 1 ml water. The cartridges were washed with 1 ml of 5% methanol in water to remove plasma proteins and the drugs were eluted with 1 ml methanol. This extraction protocol removed interferences, provided a clean background and the obtained drug recoveries were high and reproducible.
3.2 | Method validation
In order to ensure the reliability and reproducibility of the developed bioanalytical method, validation was conducted according to the guidelines of the European Medicines Agency (2012).
3.2.1 | Linearity and lower limit of quantitation
Calibration curves for SAC, VAL, ESO, CLT and NEB were constructed by plotting the peak area ratio of the drug to the IS vs. the concentration of the drug. The constructed calibration curves were linear and precise over the linearity ranges of 30–2,000 ng/ml for SAC, 70–2,000 ng/ml for VAL, ESO and CLT and 70–5,000 pg/ml for NEB. The LLOQ was 30 ng/ml (lowest standard level) with an accuracy of 113.69% for SAC, 70 ng/ml with accuracies of 98.20, 117.32 and 109.49% for VAL, ESO and CLT, respectively, and 70 pg/ml with an accuracy of 114.46% for NEB. The regression equations were also computed and the correlation coefficients were 0.9986, 0.995, 0.999, 0.991 and 0.9989 for SAC, VAL, ESO, CLT and NEB, respectively. Back calculations were conducted from the calibration curve to determine the accuracy of each calibration standard, Table 1.
3.2.2 | Selectivity
Figure 2a confirms the selectivity of the method after the analysis of the six different batches of drug-free human plasma used for analysis. There was no significant interference from matrix endogenous substances at the retention times of SAC, VAL, ESO, CLT, NEB and IS.
3.2.3 | Intra- and interday precision and accuracy
As revealed in Table 2, good results were obtained in terms of CV and recoveries for each of the cited drugs for intra- and interday precision. As per the acceptance criteria for the evaluation of precision, the deviation of each concentration level from the nominal concentration was within ±15%. Similarly, the mean accuracy did not deviate by more than ±15% of the nominal concentration (European Medicines Agency, 2012).
3.2.4 | Matrix effect
In studying the matrix effect, the MF was calculated for each drug-free human plasma matrix using six samples for the five drugs and IS. Both the MF and the IS-normalized MF were calculated. The values for CV of the IS-normalized MF calculated from the six human plasma matrix samples for the LQC and HQC samples are represented in Table 3. The results suggest that the matrix had no significant ion suppression or enhancement effects on the drugs and no significant effect on the quantification of SAC, VAL, ESO, CLT and NEB as a whole.
3.2.5 | Dilution integrity test
The dilution integrity test was evaluated by measuring the concentration of each drug in human plasma following 2- and 4-fold dilution of 4,000 ng/ml for SAC, VAL, ESO and CLT and 10,000 pg/ml for NEB. Samples showed good results concerning accuracy and precision. The values for CV for the 2-fold dilution test were 2.3, 6.9, 4.6, 7.2 and 10.6% and the accuracy results were 104.1, 101.9, 99.3, 103.2 and 103.9% for SAC, VAL, ESO, CLT and NEB, respectively. The values of CV for the 4-fold dilution test were 2.2, 5.1, 6.4, 10.0 and 11.9% and the accuracy results were 99.9, 99.1, 98.7, 99.6 and 97.0% for SAC, VAL, ESO, CLT and NEB, respectively. These results indicate that dilution of concentrated samples with blank plasma had no effect on the precision and accuracy of the method.
3.2.6 | Stability
All samples were considered stable if the deviation from the nominal concentration was within ±15%. In the short-term stability study, all of the drugs were found to be stable for 6 h in human plasma at room temperature (23–30C). In addition, they were found to be stable at 80 ± 2C in the deep freezer for 15 days in human plasma. In the autosampler, reconstituted samples of all of the drugs were stable for 24 h after sample processing. Frozen plasma samples were found to be stable even after subjecting them to three freeze–thaw cycles at concentrations corresponding to those of LQC and HQC samples (Table 4).
4 | CONCLUSION
An accurate and precise method was developed BAY 2416964 for the simultaneous estimation of a novel SAC/VAL combination, used for treatment of heart failure patients, and the co-administered drugs CLT, ESO and NEB in human plasma using UPLC–MS/MS. For effective extraction of the drugs from plasma, SPE was employed using HLB cartridges that allowed superior extraction of drugs of various polarities. To avoid the use of two ISs, a combination of two fragments of the same IS was used. Satisfactory results were obtained for validation parameters, including linearity, accuracy, precision, matrix effect, dilution integrity test and stability. The obtained results confirm the suitability of the developed method for dose adjustment in heart failure patients, in addition to bioavailability and drug–drug interaction studies.
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