Research article Received: 12 January 2015,
Revised: 15 March 2015,
Accepted: 5 June 2015
Published online in Wiley Online Library: 7 July 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3537
Simultaneous quantification of atenolol and chlorthalidone in human plasma by ultraperformance liquid chromatography–tandem mass spectrometry Jaivik V. Shaha, Daxesh P. Patelb, Priyanka A. Shaha, Mallika Sanyalc and Pranav S. Shrivastava* ABSTRACT: A simple, sensitive and reproducible ultra-performance liquid chromatography–tandem mass spectrometry method has been developed for the simultaneous determination of atenolol, a β-adrenergic receptor-blocker and chlorthalidone, a monosulfonamyl diuretic in human plasma, using atenolol-d7 and chlorthalidone-d4 as the internal standards (ISs). Following solid-phase extraction on Phenomenex Strata-X cartridges using 100 μL human plasma sample, the analytes and ISs were separated on an Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column using a mobile phase consisting of 0.1% formic acid– acetonitrile (25:75, v/v). A tandem mass spectrometer equipped with electrospray ionization was used as a detector in the positive ionization mode for both analytes. The linear concentration range was established as 0.50–500 ng/mL for atenolol and 0.25–150 ng/mL for chlorthalidone. Extraction recoveries were within 95–103% and ion suppression/enhancement, expressed as IS-normalized matrix factors, ranged from 0.95 to 1.06 for both the analytes. Intra-batch and inter-batch precision (CV) and accuracy values were 2.37–5.91 and 96.1–103.2%, respectively. Stability of analytes in plasma was evaluated under different conditions, such as bench-top, freeze–thaw, dry and wet extract and long-term. The developed method was superior to the existing methods for the simultaneous determination of atenolol and chlorthalidone in human plasma with respect to the sensitivity, chromatographic analysis time and plasma volume for processing. Further, it was successfully applied to support a bioequivalence study of 50 mg atenolol + 12.5 mg chlorthalidone in 28 healthy Indian subjects. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: atenolol; chlorthalidone; ultra-performance liquid chromatography-tandem mass spectrometry; human plasma; sensitive; bioequivalence
Introduction
208
Hypertension is one of the leading causes for cardiovascular diseases worldwide. Several mono and combination therapies are recommended for the treatment and management of mild to moderate and acute cases of hypertension. Nevertheless, majority of the patients require two or more antihypertensive agents to achieve the target blood pressure of 90% of that absorbed reaches the systemic circulation unaltered. The plasma half-life is about 6–7 h and protein binding is very low (6–16%) (Sweetman, 2002; Shrivastav et al., 2010). Chlorthalidone (CHL) is a monosulfonamyl diuretic, which increases the excretion of sodium and chloride. Although the mechanism of action for reducing blood pressure is not fully known, it is related to the excretion and redistribution of body sodium. Like ATN, CHL is also not completely absorbed (~60%) after oral administration. The plasma
* Correspondence to: P. S. Shrivastav, Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad, 380009, India. Email: [email protected] a
Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad 380009, India
b
Cancer Metastasis Alert and Prevention Centre, Fuzhou University, Fujian 350002, China
c
Department of Chemistry, St Xavier’s College, Navrangpura, Ahmedabad 380009, India Abbreviations used: ATN, atenolol; CHL, chlorthalidone; CS, calibration curve standard; MF, matrix factor.
Copyright © 2015 John Wiley & Sons, Ltd.
Determination of atenolol and chlorthalidone in plasma protein binding of CHL is ~75% and the peak plasma concentration is estimated to be in the range of 8–12 h. The plasma half-life of a single dose study varies from 22 to 55 h and the kidney is the major route of elimination (Carter et al., 2004; Pareek et al., 2008; TENORETIC®, .). The literature presents several methods for the determination of ATN, either alone (Arias et al., 2001; Chiap et al., 2000; Damiani, 2011; D’Orazio and Fanali, 2006; Iha et al., 2002; Lawson et al., 2012; Leite et al., 2006; Li et al., 2005; Spanakis and Niopas, 2013; Yilmaz et al., 2012) or with other antihypertensive agents (Bhatia et al., 2012; Braza et al., 2000; Johnson and Lewis, 2006) in biological matrices by fluorescence spectrometry (Damiani, 2011), capillary zone electrophoresis (Arias et al. 2001), liquid chromatography with UV (Bhatia et al., 2012; Chiap et al., 2000), fluorescence (Braza et al., 2000; Iha et al., 2002; Leite et al., 2006; Spanakis and Niopas, 2013; Yilmaz et al., 2012) and mass spectrometric detection (D’Orazio and Fanali, 2006; Johnson and Lewis, 2006; Lawson et al., 2012; Li et al., 2005). Similarly, CHL has been determined in human plasma, whole blood and urine by GC with nitrogen detection (Feleuren and Rossum, 1978) and HPLC-UV (Guelen et al., 1980; Muirhead and Christie, 1987; Rosenberg et al., 1986; Salado and Vera-Avila, 1997) methods. A method to determine CHL and celiprolol, a cardioselective β-blocker in tablets, human plasma and urine has also been reported (Belal et al., 2012). However, simultaneous determination of ATN and CHL is a subject of few reports (Elgawish et al., 2011; El-Glindy et al., 2008; Giachetti et al., 1997; Gonzalez et al., 2010; Khuroo et al., 2008). Amongst these, three are based on HPLC-UV/fluorescence (Elgawish et al., 2011; El-Glindy et al., 2008; Giachetti et al., 1997) analysis from human breast milk (El-Glindy et al., 2008) and plasma (Elgawish et al., 2011; Giachetti et al., 1997), while the remaining two employed LC-MS/MS technique for their simultaneous determination from human plasma (Gonzalez et al., 2010; Khuroo et al., 2008). Nevertheless, there is no UPLC-MS/MS-based method for their simultaneous estimation in any biological fluid. UPLC with 1.7 μm particle sizes has demonstrated enhanced efficiency, superior resolution, higher sensitivity and much faster throughput compared with conventional HPLC with 3 or 5 μm particles. Thus, in this communication we report a simple and precise UPLC-MS/MS method in the positive ionization mode for both the analytes. The present method is highly sensitive, rapid and rugged compared with the existing methods for these two drugs.
Experimental Chemicals and materials The reference standards and other materials used in the study are presented in the Supporting Information.
Liquid chromatographic and mass spectrometric conditions
Biomed. Chromatogr. 2016; 30: 208–216
The detail of stock solutions of analytes and ISs is presented in the Supporting Information. Calibration curve standards (CSs) were made at the following concentrations: 0.500, 1.000, 2.500, 5.000, 10.00, 20.00, 50.00, 100.0, 250.0, 500.0 ng/mL and 0.250, 0.500, 1.000, 2.500, 5.000, 10.00, 20.00, 40.00, 75.00 and 150.0 ng/mL for ATN and CHL, respectively. The quality control (QC) samples were prepared at five levels for ATN/CHL respectively: 400.0/120.0 ng/mL (HQC, high quality control); 200.0/90.00 and 25.00/15.00 ng/mL (MQC-1 and 2, medium quality control); 1.500/ 0.750 ng/mL (LQC, low quality control); and 0.500/0.250 ng/mL (LLOQ QC, lower limit of quantification quality control).
Protocol for sample preparation Prior to analysis, all frozen subject samples, CSs and QC samples were thawed and allowed to equilibrate at room temperature. To an aliquot of 100 μL of spiked plasma sample/subject sample, 25 μL (1000 ng/mL for ATN-d7 and 300 ng/mL CHL-d4) of ISs was added and vortexed for 10 s. Further, 100 μL of water was added in each tube and again vortexed for 30 s. 3 Samples were then loaded on Phenomenex Strata-X (30 mg, 1 cm ) cartridges, after conditioning with 1.0 mL methanol followed by 1 mL of water. Washing of sorbents was done with 1.0 mL of water. Subsequently, the car5 tridges were dried for 1 min by applying nitrogen (1.72 × 10 Pa) at 2.4 L/ min flow rate. Elution of analytes and ISs was done using 250 μL of mobile phase into pre-labeled vials; it was briefly vortexed for 15 s and 10 μL was used for injection in the chromatographic system.
Procedures for method validation The method was validated as per the current regulatory requirements to establish the accuracy and precision of the method (US Food and Drug Administration, 2001). The details of the parameters studied were similar to our previous report (Sharma et al., 2013) and are briefly described in the Supporting Information.
Bioequivalence study and incurred sample reanalysis In a randomized, open-label, crossover study, 28 healthy Indian subjects were administered a single fixed dose of a test (50 mg atenolol + 12.5 mg chlorthalidone tablets from a Generic Company) and a reference (TENORETIC®, 50 mg atenolol + 12.5 mg chlorthalidone tablets from AstraZeneca, Luton, UK) formulation under fasting. The study was conducted strictly in accordance with International Conference on Harmonization, E6 Good Clinical Practice guidelines (US Food and Drug Administration, 1996). The experimental details are provided in the Supporting Information. The pharmacokinetic parameters of ATN and CHL were estimated using a noncompartmental model using WinNonlin software version 5.2.1 (Pharsight Corporation, Sunnyvale, CA, USA). Assay reproducibility was also checked by reanalysing 115 subject samples and the results were compared with the initial study results. According to the acceptance criterion, at least two-thirds of the original and reanalyzed results should be within 20% of each other (Yadav and Shrivastav, 2011).
Results and discussion Method development As reported earlier, ATN has been quantified in the positive ionization mode and CHL in the negative or positive ionization mode for their simultaneous determination (Gonzalez et al., 2010; Khuroo et al., 2008). Thus, initial experiments for optimization of mass parameters were carried out in the positive as well as negative ionization mode using electrospray ionization. The response obtained for ATN was higher in the positive ionization
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
209
The chromatographic analysis of both the analytes and their ISs was performed on a Waters Acquity UPLC-type BEH C18 (50 × 2.1 mm, 1.7 μm) column and maintained at 30 °C in a column oven. The mobile phase consisted of 0.1% formic acid–acetonitrile (25:75, v/v) and was delivered at a flow rate of 0.300 mL/min. Ionization and detection of the analytes and ISs were carried out on a Waters Quattro Premier XE (USA) triple quadrupole mass spectrometer (Milford, MA, USA), equipped with electrospray ionization and operating in positive ionization mode. The optimized source dependent and compound dependent mass parameters are detailed in the Supporting Information.
Preparation of standard stock, calibration standards and quality control samples
J. V. Shah et al. mode while it was comparable in both modes for CHL. Thus, both of the analytes were ionized in the positive mode to avoid polarity switch, which requires certain time for stabilization of high voltages. The Q1 MS spectra provided predominant protonated precursor [M + H]+ ions at m/z 267.2, 274.2, 339.1 and 343.2 for ATN, ATN-d7, CHL and CHL-d4, respectively. The most consistent and intense product ions in Q3 MS were found at m/z 145.0, 145.1, 322.0 and 326.0 for ATN, ATN-d7, CHL and CHL-d4, respectively, as shown in Fig. 1(a–d). Additionally, one qualifier ion was also monitored for mass spectral identity of the analytes, m/z 267.2 → 133.0 for ATN and m/z 339.1 → 243.1 for CHL. The dwell time was kept at 100 ms, which afforded at least 26 data points across the peaks for quantitative measurements with desired sensitivity and at the same time avoiding any cross talk between ATN and ATN-d7 with identical product ions. In order to optimize the chromatographic conditions for the simultaneous determination of ATN and CHL, the effects of several chromatographic parameters like buffer pH, type of organic modifier, organic modifier–buffer ratio and the flow rate were investigated. These parameters were suitably optimized for adequate retention, acceptable peak shape, resolution and response on a BEH C18 (50 × 2.1 mm, 1.7 μm) column. Various mobile phases consisting of acetonitrile–methanol as the organic diluents together with acidic buffers (formic acid–ammonium formate and acetic acid–ammonium acetate) in the pH range of 3.0–6.0 were tested. Additives like 0.1% formic acid–acetic acid along with acetonitrile–methanol were also studied to have the best chromatographic conditions with minimal matrix interference and high sensitivity. Acetonitrile was preferred over methanol as it is more volatile and hence more suited to MS detection, while formic acid–ammonium formate buffer/0.1% formic acid in the pH range of 3.0–3.5 gave a higher and reproducible response for both the analytes. Further, as expected by increasing the organic content (80–90%), the retention time decreased but the peak shape was not acceptable. Increasing
the buffer component (40–50%) provided adequate resolution between ATN and CHL but the run time was considerably longer (>4.0 min). Thus, the mobile phase composition of 25:75 (v/v) acetonitrile–buffer was tested by varying the flow rate from 0.200 to 0.350 mL/min. The best chromatographic conditions were observed with 0.1% formic acid–acetonitrile (25:75, v/v) at a flow rate of 0.300 mL/min. Both the analytes were baseline resolved within 2.0 min with adequate retention and response across CSs and QC levels. The retention time was 0.97 and 1.47 for ATN and CHL with a resolution factor of 3.1. The reinjection reproducibility (CV) of retention times for the analytes was ≤0.85% for 75 injections on the same column. The capacity factors, which describe the rate at which the analyte migrates through the column, were 1.36 and 2.58 for ATN and CHL, respectively. Deuterated ISs used in the study effectively compensated for any possible variation in the response owing to matrix components and the results obtained showed acceptable accuracy and precision for the method. Typical multiple reaction monitoring (MRM) chromatograms for blank plasma spiked with ISs, LLOQ sample and a subject sample prove the selectivity of the developed method (Fig. 2A and B). Further there was no effect of cross-talk between ATN and its deuterated IS with identical product ions and no endogenous interference in eight different blank plasma sources studied. Infusion of analytes post-column is a useful tool for visual identification of interfering peaks that may cause matrix effects. Figure S1 in the Supporting Information shows the profiles obtained by injection of extracted blank plasma after postcolumn infusion of ATN, ATN-d7, CHL and CHL-d4 solutions. The results showed no ion suppression/enhancement of signal at the retention time of the analytes and ISs. For sample preparation, all three generic extraction methods – protein precipitation (Elgawish et al., 2011; Gonzalez et al., 2010), liquid–liquid extraction (El-Glindy et al., 2008; Giachetti et al., 1997) and solid-phase extraction (Khuroo et al., 2008) – have been
210
Figure 1. Product ion mass spectra of (a) atenolol (m/z 267.2 → 145.0, scan range 40–360 amu), (b) atenolol-d7, IS (m/z 274.2 → 145.1, scan range 40–360 amu), (c) chlorthalidone (m/z 339.1 → 322.0, scan range 40–360 amu) and (d) chlorthalidone-d4, IS (m/z 343.2 → 326.0, scan range 40–360 amu) in the positive ionization mode.
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 208–216
Determination of atenolol and chlorthalidone in plasma
Figure 2. (A) Multiple reaction monitoring (MRM) ion chromatograms of (a) blank plasma spiked with atenolol-d7, (b) atenolol at LLOQ and atenolol-d7 and (c) a real subject sample at peak concentration (Cmax) after oral administration of 50 mg atenolol + 12.5 mg chlorthalidone fixed dose formulation.
Biomed. Chromatogr. 2016; 30: 208–216
method employs deuterated ISs, while general ISs were used in these reports. Apart from one method in human breast milk (El-Gindy et al., 2008), all other methods relate to the analysis of these drugs in plasma samples. The developed UPLC-MS/MS method for ATN/CHL is at least 4/20 times more sensitive than reported methods. Further, the chromatographic analysis time is also the shortest as compared with available methods. A comparative overview of these methods and the present method are summarized in Table 1. Method validation There was minimal carry-over (≤0.11% of LLOQ area) during the autosampler carryover experiment in the extracted blank plasma, injected after the highest concentration of ATN (500 ng/mL) and CHL (150 ng/mL). The best fittings of the calibration curves were obtained by linear regression with 1/x2 weighting in the concentration range of 0.500–500 ng/mL (r2, 0.9999) for ATN and 0.250–150 ng/mL (r2, 0.9998) for CHL. The mean linear equations obtained were y = (0.004957 ± 0.000014) x + (0.000126 ± 0.000012) for ATN and y = (0.010980 ± 0.000083) x + (0.000143 ± 0.000094) for CHL. The accuracy and precision
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
211
reported for these drugs. Thus, protein precipitation, being the simplest and an efficient technique for gross removal of proteins was tested with common precipitants like methanol, ethanol, acetonitrile and perchloric acid. However, the recovery was not consistent at lower QC levels, especially for ATN. Similar results were obtained by liquid–liquid extraction with different organic diluents like methyl tert-butyl ether, dichloromethane-2-propanol (El-Glindy et al., 2008) and ethyl acetate. Although the recovery of the analytes was in the range of 60–75%, it was inconsistent. Thus, solid-phase extraction, which provides cleaner extracts through selective retention of the analytes on the column phase and for effective removal of endogenous moieties in plasma, was tested on a Phenomenex Strata-X (30 mg, 1 cm3) extraction cartridge. Washing with 1.0 mL water was adequate to remove any endogenous components and elution with 0.1% formic acid–acetonitrile (25:75, v/v) provided precise and quantitative recovery for both the analytes. The developed method was superior to the existing methods for their simultaneous determination in biological fluids with respect to sensitivity, throughput and sample processing volume (Elgawish et al., 2011; El-Glindy et al., 2008; Giachetti et al., 1997; Gonzalez et al., 2010; Khuroo et al., 2008). Additionally, the present
J. V. Shah et al.
Figure 2. (Continued)
212
(CV) observed for the calibration curve standards ranged from 97.7 to 102.3 and from 1.30 to 3.77% for ATN and from 97.6 to 102.5 and from 1.16 to 2.81% for CHL, respectively. The lowest concentration (LLOQ) in the standard curve that was measured with acceptable accuracy and precision was 0.50 and 0.250 ng/mL for ATN and CHL, respectively, at a signalto-noise ratio (S/N) of ≥10. The intra- and inter-batch precision and accuracy were established at five QC levels in validation runs performed at five QC levels (Table S1 in the Supporting Information). The intra-batch precision (CV) ranged from 2.37 to 5.73 and the accuracy was within 96.1–103.2% for both the analytes. Likewise, for the interbatch experiments, the precision varied from 2.72 to 5.91 and the accuracy was within 96.1–103.0%. The extraction recovery of analytes from solid-phase extraction ranged from 95.4 to 102.7 for ATN and from 95.6 to 103.2% for CHL. The mean recovery of ATN-d7 and CHL-d4 was 98.2 and 97.8%, respectively. The matrix effect is responsible for suppression or enhancement in the measurement of analyte signal owing to endogenous or exogenous components present in biological fluids. The absolute matrix effect, expressed as matrix factor (MF) was evaluated at four QC levels. The MFs were calculated from the peak area response for the analytes and their ISs
wileyonlinelibrary.com/journal/bmc
separately and their ratios were then used to find the IS-normalized MF, which ranged from 0.95 to 1.06 across four QC levels for both of the analytes (Table 2). Further, relative matrix effect was assessed in eight different plasma sources (five normal K3EDTA, one heparinized, one haemolysed and one lipemic). The precision (CV) in the measurement of slope of calibration lines was 2.68 and 2.02 for ATN and CHL respectively as shown in Table S2 in the Supporting Information. The samples kept for short-term and long term stock and working solutions of ATN, CHL and ISs remained unaffected up to 26 h and 25 days, respectively. The bench-top stability of analytes in plasma was determined up to 18 h and for five freeze–thaw cycles. Autosampler stability of the spiked QC samples maintained at 5 °C was established up to 48 h and at ambient temperature up to 12 h without significant loss of analytes. The dry extracts kept at 20 °C were stable up to 18 h. The samples stored for assessment of longterm stability of analytes in plasma were found stable for a minimum period of 70 days. The detailed stability results are summarized in Table 3. The precision (CV) and accuracy for method ruggedness with different columns and analysts ranged from 2.74 to 4.05% and 94.3 to 101.5%, respectively, across five QC levels for both of the analytes. While the precision (CV) for dilution reliability of 1/5th
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 208–216
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
LC-MS-MS; 10–2050 for ATN and 15–3035 for CHL
LC-MS-MS; 2.0–1000 for ATN and 5.0–500 for CHL
UPLC-MS-MS; 0.5–500 for ATN and 0.25–150 for CHL
4
5a
6
Supelcosil LC18 (250 × 4 mm, 5.0 μm); ATN – 15.70 min, CHL – 6.05 min; 22.0 min Luna CN (250 × 4.6 mm, 5.0 μm); ATN – 9.5 min, CHL – 5.0 min; 12.0 min Shim-pack® CN-propyl (250 × 4.6 mm, 5.0 μm); ATN – ~5.2 min, CHL – ~8.5 min; ~12.0 min Xterra C18 LC (150 × 4.6 mm, 5.0 μm); ATN – 3.54, CHL – 2.11; 3.0 min Luna C18 (150 × 4.6 mm, 3.0 μm); ATN – 4.5, CHL – 7.2; 1 2.0 min Acquity BEH C18 (50 × 2.1 mm, 1.7 μm); ATN – 0.97, CHL – 1.47; 2.0 min
LLE; 1000 μL plasma; salbutamol and xipamide
SPE; 100 μL human plasma; atenolol-d7 and chlorthalidone-d4
PP; 500 μL human plasma; pravastatin
SPE; 300 μL human plasma; metoprolol and hydrochlorothiazide
PP; 960 μL human plasma; hydrochlorothiazide
LLE; 1000 μL breast milk; guaifenesin
Column; retention time; run time
Extraction; sample volume; internal standard
Bioequivalence study with 100 + 25 mg of ATN + CHL tablets in 23 healthy subjects Measurement of plasma concentration of ATN, CHL and other cardiovascular drugs in 14 patients Bioequivalence study with 50 + 12.5 mg of ATN + CHL tablets in 28 healthy subjects
Determination of ATN and CHL in spiked human plasma
Pharmacokinetic studies with 100 + 25 mg and 50 + 12.5 mg of ATN + CHL tablets in 18 healthy subjects Determination of atenolol and chlorthalidone in human breast milk
Application
PM
Gonzalez et al. (2010)
Khuroo et al. (2008)
Elgawish et al. (2011)
El-Gindy et al. (2008)
Giachetti et al. (1997)
Reference
a Together with other cardiovascular drugs. ATN, Atenolol; CHL, chlorthalidone; LLE, liquid–liquid extraction; SPE, solid-phase extraction; PP, protein precipitation extraction, PM, present method.
HPLC-UV; 100–10,000 for ATN and CHL
3
HPLC-UV for CHL and Fluorescence for ATN; 10–1000 for ATN and CHL
Detection technique; linearity (ng/mL)
HPLC-UV; 300–20,000 for ATN and 250–5000 for CHL
213
Biomed. Chromatogr. 2016; 30: 208–216
2
1
Serial No.
Table 1. Comparison of salient features of chromatographic methods developed for simultaneous determination of atenolol and chlorthalidone
Determination of atenolol and chlorthalidone in plasma
J. V. Shah et al. Table 2. Extraction recovery and matrix factor for atenolol and chlorthalidone QC level
Area response (replicate, n = 6) A (post-extraction spiking)
Atenolol LQC MQC-2 MQC-1 HQC Chlorthalidone LQC MQC-2 MQC-1 HQC
B (pre-extraction spiking)
C (neat samples in mobile phase)
Extraction recovery, % (B/A)
Matrix factor Analyte (A/C)
IS
ISnormalized
2,357 39,171 313,335 626,652
2,248 38,456 321,795 602,839
2,507 38,402 319,729 646,032
95.4 (95.8)a 98.2 (97.3)a 102.7 (103.1)a 96.2 (96.4)a
0.94 1.02 0.98 0.97
0.99 0.96 1.03 0.98
0.95 1.06 0.95 0.99
1,163 23,265 139,554 186,135
1,200 22,427 133,413 182,040
1,129 23,034 145,368 195,931
103.2 (102.4)b 96.4 (96.7)b 95.6 (95.1)b 97.8 (97.2)b
1.03 1.01 0.96 0.95
1.01 0.95 0.99 0.97
1.02 1.06 0.97 0.98
a
Values for atenolol-d7. values for chlorthalidone-d4. IS, Internal standard; LQC: low quality control; MQC: medium quality control; HQC: high quality control.
b
Table 3. Stability of atenolol and chlorthalidone in plasma under various conditions (replicates, n = 6) Storage conditions
Bench-top stability at room temperature, 18 h Freeze–thaw stability at 20 °C Freeze–thaw stability at 70 °C Processed sample stability room temperature, 12 h Autosampler stability at 5 °C, 48 h Dry extract stability at 5 °C, 18 h Long-term stability at 20 °C, 70 days Long-term stability at 70 °C, 70 days
Atenolol
Chlorthalidone
Nominal concentration (ng/mL)
Mean stability sample (ng/mL) ± SD
Percentage change
Nominal concentration (ng/mL)
Mean stability sample (ng/mL) ± SD
Percentage change
400.0 1.500 400.0 1.500 400.0 1.500 400.0 1.500 400.0 1.500 400.0 1.500 400.0 1.500 400.0 1.500
412.7 ± 3.32 1.561 ± 0.027 383.2 ± 5.77 1.445 ± 0.028 411.8 ± 7.47 1.578 ± 0.008 386.2 ± 4.34 1.570 ± 0.023 392.2 ± 5.22 1.592 ± 0.027 406.0 ± 4.31 1.420 ± 0.031 390.2 ± 3.80 1.409 ± 0.024 416.4 ± 4.26 1.435 ± 0.020
3.18 4.06 4.20 3.66 2.95 5.20 3.45 4.66 1.95 6.13 1.50 5.33 2.45 6.07 4.10 4.33
120.0 0.750 120.0 0.750 120.0 0.750 120.0 0.750 120.0 0.750 120.0 0.750 120.0 0.750 120.0 0.750
121.9 ± 1.26 0.707 ± 0.007 124.5 ± 1.60 0.726 ± 0.006 117.9 ± 1.96 0.782 ± 0.008 116.0 ± 2.21 0.795 ± 0.005 122.8 ± 2.30 0.773 ± 0.005 115.0 ± 1.39 0.793 ± 0.004 124.9 ± 1.04 0.717 ± 0.008 116.5 ± 1.64 0.706 ± 0.008
1.58 5.73 3.75 3.20 1.75 4.27 3.33 6.00 2.33 3.06 4.17 5.73 4.08 4.40 2.92 5.87
SD, Standard deviation; n, number of replicates. Mean stability samples – Mean comparison samples Percentage change ¼ 100 Mean comparison samples
and 1/10th dilution were between 0.96 and 3.75%, and the accuracy ranged from 96.2 to 102.9%.
214
Application of the method The aim of the study was to test the comparative bioavailability of a test and reference product under fasting and thereby to determine their bioequivalence. To minimize variability and focus
wileyonlinelibrary.com/journal/bmc
on the comparison of the two formulations, healthy volunteers were selected in the present work. The validated method was applied using a low-dose combination of ATN (50 mg) and CHL (12.5 mg) in healthy subjects for better efficacy-to-side effect ratio as reported previously (Carter et al., 2004; Pareek et al., 2008). Figure 3 shows the time course profile of ATN and CHL plasma concentration under fasting. The mean pharmacokinetic parameters evaluated after oral administration of combination tablet
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 208–216
Determination of atenolol and chlorthalidone in plasma Table 4. Mean pharmacokinetic parameters following oral administration of 50 mg atenolol + 12.5 mg chlorthalidone combination formulation in 28 healthy Indian subjects under fasting Parameter
Cmax (ng/mL) Tmax (h) t1/2 (h) AUC 0–120 (h ng/mL) AUC 0–inf (h ng/mL) Kel (1/h)
Atenolol (mean ± SD)
Chlorthalidone (mean ± SD)
Test
Reference
Test
Reference
321.6 ± 7.9 3.11 ± 0.28 7.75 ± 0.22 2387.7 ± 32.6 2507.1 ± 38.3 0.089 ± 0.018
315.2 ± 6.6 3.07 ± 0.20 7.51 ± 0.14 2316.5 ± 29.8 2432.3 ± 37.2 0.092 ± 0.014
74.8 ± 3.6 5.97 ± 0.19 31.7 ± 0.15 1048.4 ± 20.3 1093.5 ± 26.1 0.022 ± 0.003
73.3 ± 3.9 5.74 ± 0.13 31.5 ± 0.10 1019.6 ± 23.6 1066.5 ± 25.2 0.023 ± 0.003
Cmax, Maximum plasma concentration; Tmax, time point of maximum plasma concentration; t1/2, half life of drug elimination during the terminal phase; AUC0–t, area under the plasma concentration-time curve from 0 to 120 h; AUC0–inf, area under the plasma concentration-time curve from zero hour to infinity; Kel, elimination rate constant; SD, standard deviation. Reference formulation: Tenoretic®, 50 mg atenolol and 12.5 mg chlorthalidone tablets from AstraZeneca UK Limited, Luton, UK. Test formulation: 50 mg atenolol and 12.5 mg chlorthalidone tablets from a Generic Company, India.
115 study samples were selected which were near the Cmax and the elimination phase in the pharmacokinetic profile of the drugs. These samples were reanalyzed and the results were compared with the initial study results. The percentage change in the results was within ±14% for both the analytes, which is within the acceptance criterion. The graphical representation of the results is shown in Figure S2 in the Supporting Information.
Conclusion A highly sensitive, rapid and rugged UPLC-MS/MS method has been developed for the simultaneous quantitation of ATN and CHL in human plasma. Absence of matrix interference was suitably verified through IS-normalized matrix factors and by calculation of precision in the measurement of slope of calibration curves prepared in different plasma sources. The statistical treatment data reveal high accuracy and precision of the proposed method for all of the validation parameters. The method was successfully applied to measure plasma concentration of ATN and CHL in subject samples. Finally, the results of incurred sample reanalysis confirm the reproducibility of the proposed method.
Acknowledgments Figure 3. Mean plasma concentration–time profile of atenolol and chlorthalidone after oral administration of test (50 mg atenolol + 12.5 mg chlorthalidone tablets from a Generic Company) and a reference (TENORETIC®, 50 mg atenolol + 12.5 mg chlorthalidone tablets from AstraZeneca UK Limited, Luton, UK) formulation to 28 healthy subjects.
One of the authors, Jaivik V. Shah wishes to thank UGC, New Delhi for BSR Fellowship and to the management of Cadila Pharmaceuticals Ltd, Ahmedabad, India for providing instrumentation and infrastructure facility to carry out this work.
References
Biomed. Chromatogr. 2016; 30: 208–216
Arias R, Jimenez RM, Alomso RM, Telez M, Arrieta I, Flores P and Lastra EO. Determination of the β-blocker atenolol in plasma by capillary zone electrophoresis. Journal of Chromatography A 2001; 916: 297–304. Belal F, El-Brashy AM, El-Enany N and Tolba MM. Simultaneous determination of celiprolol HCl and chlorthalidone in tablets and biological fluids using high-performance liquid chromatography. Acta Chromatographica 2012; 24: 185–206. Bhatia NM, Gurav SB, Jadhav SD and Bhatia MS. RP-HPLC method for simultaneous estimation of atorvastatin calcium, losartan potassium, atenolol and aspirin from tablet dosage form and plasma. Journal of Liquid Chromatography and Related Technologies 2012; 35: 428–443.
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
215
are summarized in Table 4. The mean peak concentration (Cmax), time to reach Cmax (Tmax) and area under the concentration–time curve (AUC0–120 h) were 321.6 ± 7.9 ng/mL, 3.11 h and 2387.7 h. ng/mL for ATN and 74.8 ng/mL, 5.97 h and 1048.4 h. ng/mL for CHL, respectively. These values were in agreement with a previous method with healthy Indian subjects based on dose strength (Khuroo et al., 2008). Further, CHL had a long half-life of 31.7 h compared with 7.75 h for ATN as described in several reports (TENORETIC®, ). To prove the method’s reproducibility,
J. V. Shah et al. Braza AJ, Modamio P and Marino EL. Two reproducible and sensitive liquid chromatographic methods to quantify atenolol and propranolol in human plasma and determination of their associated analytical error functions. Journal of Chromatography B 2000; 738: 225–231. Carter BL, Ernst ME and Cohen JD. Hydrochlorothiazide versus chlorthalidone: evidence supporting their interchangeability. Hypertension 2004; 43: 4–9. Chiap P, Buraglia M, Ceccato A, Hubert P and Crommen J. Automated liquid chromatographic determination of atenolol in plasma using dialysis and trace enrichment on a cation-exchange precolumn for sample handling. Journal of Chromatography B 2000; 739: 205–217. Damiani PC. Determination of atenolol in human urine by emissionexcitation fluorescence matrices and unfolded partial least-squares with residual bilinearization. Talanta 2011; 85: 1526–1534. D’Orazio G and Fanali S. Use of tecicoplanin stationary phase for the enantiomeric resolution of atenolol in human urine by nano-liquid chromatography–mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2006; 40: 539–544. Elgawish MS, Mostafa SM and Elshanawane AA. Simple and rapid HPLC method for simultaneous determination of atenolol and chlorthalidone in spiked human plasma. Saudi Pharmaceutical Journal 2011; 19: 43–49. El-Glindy A, Sallam S and Abdel-Salam RA. HPLC method for the simultaneous determination of atenolol and chlorthalidone in human breat milk. Journal of Separation Science 2008; 31: 677–682. Feleuren HLJM and Rossum JMV. Determination of chlorthalidone in plasma, urine and red blood cells by gas chromatography with nitrogen detection. Journal of Chromatography 1978; 152: 41–54. Giachetti C, Tenconi A, Canali S and Zanolo G. Simultaneous determination of atenolol and chlorthalidone in plasma by high-performance liquid chromatography. Application to pharmacokinetic studies in man. Journal of Chromatography B 1997; 698: 187–194. Gonzalez O, Iriarte G, Rico E, Ferreiros N, Maguregui MI, Alonso RM and Jimenez RM. LC-MS/MS method for the determination of several drugs used in combined cardiovascular therapy in human plasma. Journal of Chromatography B 2010; 878: 2685–2692. Guelen PJM, Baars AM and Vree TB. Rapid and sensitive determination of chlorthalidone in blood, plasma and urine of man using highperformance liquid chromatography. Journal of Chromatography 1980; 181: 497–503. Iha MH, Martinez AS and Bonato PS. Enantioselective analysis of atenolol in biologic fluids: comparison of liquid–liquid and solid-phase extraction methods. Journal of Chromatography B 2002; 767: 1–9. Johnson RD and Lewis RJ. Quantification of atenolol, metraprolol, and propanolol in postmortem human fluid and tissue specimens via LC/APCI-MS. Forensic Science International 2006; 156: 106–117. Khuroo A, Mishra S, Singh O, Saxena S and Monif T. Simultaneous determination of atenolol and chlorthalidone by LC-MS-MS in human plasma. Chromatographia 2008; 68: 721–729. Lawson G, Cocks E and Tanna S. Quantitative determination of atenolol in dried blood spot samples by LC-HRMS: a potential method for assessing medication adherance. Journal of Chromatography B 2012; 897: 72–79. Leite FS, Pereira VA, Omosako CE, Carmona MJC, Auler JOC and Santos SRCJ. A micromethod for the quantification of atenolol in plasma using high-performance liquid chromatography with fluorescence detection. Therapeutic Drug Monitoring 2006; 28: 237–244.
Li W, Li Y, Francisco DT and Naidong W. Hydrophilic interaction liquid chromatographic tandem mass spectrometric determination of atenolol in human plasma. Biomedical Chromatography 2005; 19: 385–393. Muirhead DC and Christie RB. Simple, sensitive and selective highperformance liquid chromatographic method for analysis of chlorthaidone in whole blood. Journal of Chromatography 1987; 416: 420–425. Pareek A, Karnik N, Salagre SB, Zawar SD, Joglekar VK, Chandurkar N and Naik GS. Clinical effectiveness of low-dose chlorthalidone (6.25 mg) + atenolol combination in stage I hypertensive patients: a multicenter, randomized, controlled study. Current Medical Research and Opinion 2008; 24: 1771–1779. Rosenberg MJ, Lam KK and Dorsey TE. Analysis of chlorthaidone in whole blodd by high-performance liquid chromatography. Journal of Chromatography 1986; 375: 438–443. Salado SC and Vera-Avila LE. On-line solid-phase extraction and highperformance liquid chromatographic determination of chlorthalidone in urine. Journal of Chromatography B 1997; 690: 195–202. Sharma P, Patel DP, Sanyal M, Berawala H, Guttikar S and Shrivastav PS. Simultaneous analysis of oxybutynin and its active metabolite N-desethyl oxybutynin in human plasma by stable isotope dilution LC-MS/MS to support a bioequivalence study. Journal of Pharmaceutical and Biomedical Analysis 2013; 84: 244–255. Shrivastav PS, Buha SM and Sanyal M. Detection and quantitation of betablockers in plasma and urine. Bioanalysis 2010; 2: 263–276. Spanakis M and Niopas I. Determination of atenolol in human plasma by HPLC with fluorescence detection: validation and application in a pharmacokinetic study. Journal of Chromatographic Science 2013; 52: 128–132. Sweetman SC. , 33rd edn, Martindale – The Complete Drug Reference. Pharmaceutical Press, Royal Pharmaceutical Society of Great Britain: London , 2002. TENORETIC® (atenolol and chlorthalidone). Product details. AstraZeneca Pharmaceuticals, London, n.d. Available from: http://www1. astrazeneca-us.com/pi/tenoretic_lpv.pdf (accessed December 2014). US Food and Drug Administration. Guidance for Industry: ICH E6 Good Clinical Practice. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research and Centre for Biologics Evaluation and Research, 1996. US Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation. U.S. Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research and Centre for Veterinary Medicine, 2001. Yadav M and Shrivastav PS. Incurred sample reanalysis: a decisive tool in bioanalytical research. Bioanalysis 2011; 3: 1007–1024. Yilmaz B, Arslan S and Asci A. HPLC method for determination of atenolol in human plasma and application to a pharmacokinetic study in Turkey. Journal of Chromatographic Science 2012; 50: 914–919.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
216 wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 208–216
Revised: 15 March 2015,
Accepted: 5 June 2015
Published online in Wiley Online Library: 7 July 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3537
Simultaneous quantification of atenolol and chlorthalidone in human plasma by ultraperformance liquid chromatography–tandem mass spectrometry Jaivik V. Shaha, Daxesh P. Patelb, Priyanka A. Shaha, Mallika Sanyalc and Pranav S. Shrivastava* ABSTRACT: A simple, sensitive and reproducible ultra-performance liquid chromatography–tandem mass spectrometry method has been developed for the simultaneous determination of atenolol, a β-adrenergic receptor-blocker and chlorthalidone, a monosulfonamyl diuretic in human plasma, using atenolol-d7 and chlorthalidone-d4 as the internal standards (ISs). Following solid-phase extraction on Phenomenex Strata-X cartridges using 100 μL human plasma sample, the analytes and ISs were separated on an Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column using a mobile phase consisting of 0.1% formic acid– acetonitrile (25:75, v/v). A tandem mass spectrometer equipped with electrospray ionization was used as a detector in the positive ionization mode for both analytes. The linear concentration range was established as 0.50–500 ng/mL for atenolol and 0.25–150 ng/mL for chlorthalidone. Extraction recoveries were within 95–103% and ion suppression/enhancement, expressed as IS-normalized matrix factors, ranged from 0.95 to 1.06 for both the analytes. Intra-batch and inter-batch precision (CV) and accuracy values were 2.37–5.91 and 96.1–103.2%, respectively. Stability of analytes in plasma was evaluated under different conditions, such as bench-top, freeze–thaw, dry and wet extract and long-term. The developed method was superior to the existing methods for the simultaneous determination of atenolol and chlorthalidone in human plasma with respect to the sensitivity, chromatographic analysis time and plasma volume for processing. Further, it was successfully applied to support a bioequivalence study of 50 mg atenolol + 12.5 mg chlorthalidone in 28 healthy Indian subjects. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: atenolol; chlorthalidone; ultra-performance liquid chromatography-tandem mass spectrometry; human plasma; sensitive; bioequivalence
Introduction
208
Hypertension is one of the leading causes for cardiovascular diseases worldwide. Several mono and combination therapies are recommended for the treatment and management of mild to moderate and acute cases of hypertension. Nevertheless, majority of the patients require two or more antihypertensive agents to achieve the target blood pressure of 90% of that absorbed reaches the systemic circulation unaltered. The plasma half-life is about 6–7 h and protein binding is very low (6–16%) (Sweetman, 2002; Shrivastav et al., 2010). Chlorthalidone (CHL) is a monosulfonamyl diuretic, which increases the excretion of sodium and chloride. Although the mechanism of action for reducing blood pressure is not fully known, it is related to the excretion and redistribution of body sodium. Like ATN, CHL is also not completely absorbed (~60%) after oral administration. The plasma
* Correspondence to: P. S. Shrivastav, Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad, 380009, India. Email: [email protected] a
Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad 380009, India
b
Cancer Metastasis Alert and Prevention Centre, Fuzhou University, Fujian 350002, China
c
Department of Chemistry, St Xavier’s College, Navrangpura, Ahmedabad 380009, India Abbreviations used: ATN, atenolol; CHL, chlorthalidone; CS, calibration curve standard; MF, matrix factor.
Copyright © 2015 John Wiley & Sons, Ltd.
Determination of atenolol and chlorthalidone in plasma protein binding of CHL is ~75% and the peak plasma concentration is estimated to be in the range of 8–12 h. The plasma half-life of a single dose study varies from 22 to 55 h and the kidney is the major route of elimination (Carter et al., 2004; Pareek et al., 2008; TENORETIC®, .). The literature presents several methods for the determination of ATN, either alone (Arias et al., 2001; Chiap et al., 2000; Damiani, 2011; D’Orazio and Fanali, 2006; Iha et al., 2002; Lawson et al., 2012; Leite et al., 2006; Li et al., 2005; Spanakis and Niopas, 2013; Yilmaz et al., 2012) or with other antihypertensive agents (Bhatia et al., 2012; Braza et al., 2000; Johnson and Lewis, 2006) in biological matrices by fluorescence spectrometry (Damiani, 2011), capillary zone electrophoresis (Arias et al. 2001), liquid chromatography with UV (Bhatia et al., 2012; Chiap et al., 2000), fluorescence (Braza et al., 2000; Iha et al., 2002; Leite et al., 2006; Spanakis and Niopas, 2013; Yilmaz et al., 2012) and mass spectrometric detection (D’Orazio and Fanali, 2006; Johnson and Lewis, 2006; Lawson et al., 2012; Li et al., 2005). Similarly, CHL has been determined in human plasma, whole blood and urine by GC with nitrogen detection (Feleuren and Rossum, 1978) and HPLC-UV (Guelen et al., 1980; Muirhead and Christie, 1987; Rosenberg et al., 1986; Salado and Vera-Avila, 1997) methods. A method to determine CHL and celiprolol, a cardioselective β-blocker in tablets, human plasma and urine has also been reported (Belal et al., 2012). However, simultaneous determination of ATN and CHL is a subject of few reports (Elgawish et al., 2011; El-Glindy et al., 2008; Giachetti et al., 1997; Gonzalez et al., 2010; Khuroo et al., 2008). Amongst these, three are based on HPLC-UV/fluorescence (Elgawish et al., 2011; El-Glindy et al., 2008; Giachetti et al., 1997) analysis from human breast milk (El-Glindy et al., 2008) and plasma (Elgawish et al., 2011; Giachetti et al., 1997), while the remaining two employed LC-MS/MS technique for their simultaneous determination from human plasma (Gonzalez et al., 2010; Khuroo et al., 2008). Nevertheless, there is no UPLC-MS/MS-based method for their simultaneous estimation in any biological fluid. UPLC with 1.7 μm particle sizes has demonstrated enhanced efficiency, superior resolution, higher sensitivity and much faster throughput compared with conventional HPLC with 3 or 5 μm particles. Thus, in this communication we report a simple and precise UPLC-MS/MS method in the positive ionization mode for both the analytes. The present method is highly sensitive, rapid and rugged compared with the existing methods for these two drugs.
Experimental Chemicals and materials The reference standards and other materials used in the study are presented in the Supporting Information.
Liquid chromatographic and mass spectrometric conditions
Biomed. Chromatogr. 2016; 30: 208–216
The detail of stock solutions of analytes and ISs is presented in the Supporting Information. Calibration curve standards (CSs) were made at the following concentrations: 0.500, 1.000, 2.500, 5.000, 10.00, 20.00, 50.00, 100.0, 250.0, 500.0 ng/mL and 0.250, 0.500, 1.000, 2.500, 5.000, 10.00, 20.00, 40.00, 75.00 and 150.0 ng/mL for ATN and CHL, respectively. The quality control (QC) samples were prepared at five levels for ATN/CHL respectively: 400.0/120.0 ng/mL (HQC, high quality control); 200.0/90.00 and 25.00/15.00 ng/mL (MQC-1 and 2, medium quality control); 1.500/ 0.750 ng/mL (LQC, low quality control); and 0.500/0.250 ng/mL (LLOQ QC, lower limit of quantification quality control).
Protocol for sample preparation Prior to analysis, all frozen subject samples, CSs and QC samples were thawed and allowed to equilibrate at room temperature. To an aliquot of 100 μL of spiked plasma sample/subject sample, 25 μL (1000 ng/mL for ATN-d7 and 300 ng/mL CHL-d4) of ISs was added and vortexed for 10 s. Further, 100 μL of water was added in each tube and again vortexed for 30 s. 3 Samples were then loaded on Phenomenex Strata-X (30 mg, 1 cm ) cartridges, after conditioning with 1.0 mL methanol followed by 1 mL of water. Washing of sorbents was done with 1.0 mL of water. Subsequently, the car5 tridges were dried for 1 min by applying nitrogen (1.72 × 10 Pa) at 2.4 L/ min flow rate. Elution of analytes and ISs was done using 250 μL of mobile phase into pre-labeled vials; it was briefly vortexed for 15 s and 10 μL was used for injection in the chromatographic system.
Procedures for method validation The method was validated as per the current regulatory requirements to establish the accuracy and precision of the method (US Food and Drug Administration, 2001). The details of the parameters studied were similar to our previous report (Sharma et al., 2013) and are briefly described in the Supporting Information.
Bioequivalence study and incurred sample reanalysis In a randomized, open-label, crossover study, 28 healthy Indian subjects were administered a single fixed dose of a test (50 mg atenolol + 12.5 mg chlorthalidone tablets from a Generic Company) and a reference (TENORETIC®, 50 mg atenolol + 12.5 mg chlorthalidone tablets from AstraZeneca, Luton, UK) formulation under fasting. The study was conducted strictly in accordance with International Conference on Harmonization, E6 Good Clinical Practice guidelines (US Food and Drug Administration, 1996). The experimental details are provided in the Supporting Information. The pharmacokinetic parameters of ATN and CHL were estimated using a noncompartmental model using WinNonlin software version 5.2.1 (Pharsight Corporation, Sunnyvale, CA, USA). Assay reproducibility was also checked by reanalysing 115 subject samples and the results were compared with the initial study results. According to the acceptance criterion, at least two-thirds of the original and reanalyzed results should be within 20% of each other (Yadav and Shrivastav, 2011).
Results and discussion Method development As reported earlier, ATN has been quantified in the positive ionization mode and CHL in the negative or positive ionization mode for their simultaneous determination (Gonzalez et al., 2010; Khuroo et al., 2008). Thus, initial experiments for optimization of mass parameters were carried out in the positive as well as negative ionization mode using electrospray ionization. The response obtained for ATN was higher in the positive ionization
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
209
The chromatographic analysis of both the analytes and their ISs was performed on a Waters Acquity UPLC-type BEH C18 (50 × 2.1 mm, 1.7 μm) column and maintained at 30 °C in a column oven. The mobile phase consisted of 0.1% formic acid–acetonitrile (25:75, v/v) and was delivered at a flow rate of 0.300 mL/min. Ionization and detection of the analytes and ISs were carried out on a Waters Quattro Premier XE (USA) triple quadrupole mass spectrometer (Milford, MA, USA), equipped with electrospray ionization and operating in positive ionization mode. The optimized source dependent and compound dependent mass parameters are detailed in the Supporting Information.
Preparation of standard stock, calibration standards and quality control samples
J. V. Shah et al. mode while it was comparable in both modes for CHL. Thus, both of the analytes were ionized in the positive mode to avoid polarity switch, which requires certain time for stabilization of high voltages. The Q1 MS spectra provided predominant protonated precursor [M + H]+ ions at m/z 267.2, 274.2, 339.1 and 343.2 for ATN, ATN-d7, CHL and CHL-d4, respectively. The most consistent and intense product ions in Q3 MS were found at m/z 145.0, 145.1, 322.0 and 326.0 for ATN, ATN-d7, CHL and CHL-d4, respectively, as shown in Fig. 1(a–d). Additionally, one qualifier ion was also monitored for mass spectral identity of the analytes, m/z 267.2 → 133.0 for ATN and m/z 339.1 → 243.1 for CHL. The dwell time was kept at 100 ms, which afforded at least 26 data points across the peaks for quantitative measurements with desired sensitivity and at the same time avoiding any cross talk between ATN and ATN-d7 with identical product ions. In order to optimize the chromatographic conditions for the simultaneous determination of ATN and CHL, the effects of several chromatographic parameters like buffer pH, type of organic modifier, organic modifier–buffer ratio and the flow rate were investigated. These parameters were suitably optimized for adequate retention, acceptable peak shape, resolution and response on a BEH C18 (50 × 2.1 mm, 1.7 μm) column. Various mobile phases consisting of acetonitrile–methanol as the organic diluents together with acidic buffers (formic acid–ammonium formate and acetic acid–ammonium acetate) in the pH range of 3.0–6.0 were tested. Additives like 0.1% formic acid–acetic acid along with acetonitrile–methanol were also studied to have the best chromatographic conditions with minimal matrix interference and high sensitivity. Acetonitrile was preferred over methanol as it is more volatile and hence more suited to MS detection, while formic acid–ammonium formate buffer/0.1% formic acid in the pH range of 3.0–3.5 gave a higher and reproducible response for both the analytes. Further, as expected by increasing the organic content (80–90%), the retention time decreased but the peak shape was not acceptable. Increasing
the buffer component (40–50%) provided adequate resolution between ATN and CHL but the run time was considerably longer (>4.0 min). Thus, the mobile phase composition of 25:75 (v/v) acetonitrile–buffer was tested by varying the flow rate from 0.200 to 0.350 mL/min. The best chromatographic conditions were observed with 0.1% formic acid–acetonitrile (25:75, v/v) at a flow rate of 0.300 mL/min. Both the analytes were baseline resolved within 2.0 min with adequate retention and response across CSs and QC levels. The retention time was 0.97 and 1.47 for ATN and CHL with a resolution factor of 3.1. The reinjection reproducibility (CV) of retention times for the analytes was ≤0.85% for 75 injections on the same column. The capacity factors, which describe the rate at which the analyte migrates through the column, were 1.36 and 2.58 for ATN and CHL, respectively. Deuterated ISs used in the study effectively compensated for any possible variation in the response owing to matrix components and the results obtained showed acceptable accuracy and precision for the method. Typical multiple reaction monitoring (MRM) chromatograms for blank plasma spiked with ISs, LLOQ sample and a subject sample prove the selectivity of the developed method (Fig. 2A and B). Further there was no effect of cross-talk between ATN and its deuterated IS with identical product ions and no endogenous interference in eight different blank plasma sources studied. Infusion of analytes post-column is a useful tool for visual identification of interfering peaks that may cause matrix effects. Figure S1 in the Supporting Information shows the profiles obtained by injection of extracted blank plasma after postcolumn infusion of ATN, ATN-d7, CHL and CHL-d4 solutions. The results showed no ion suppression/enhancement of signal at the retention time of the analytes and ISs. For sample preparation, all three generic extraction methods – protein precipitation (Elgawish et al., 2011; Gonzalez et al., 2010), liquid–liquid extraction (El-Glindy et al., 2008; Giachetti et al., 1997) and solid-phase extraction (Khuroo et al., 2008) – have been
210
Figure 1. Product ion mass spectra of (a) atenolol (m/z 267.2 → 145.0, scan range 40–360 amu), (b) atenolol-d7, IS (m/z 274.2 → 145.1, scan range 40–360 amu), (c) chlorthalidone (m/z 339.1 → 322.0, scan range 40–360 amu) and (d) chlorthalidone-d4, IS (m/z 343.2 → 326.0, scan range 40–360 amu) in the positive ionization mode.
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 208–216
Determination of atenolol and chlorthalidone in plasma
Figure 2. (A) Multiple reaction monitoring (MRM) ion chromatograms of (a) blank plasma spiked with atenolol-d7, (b) atenolol at LLOQ and atenolol-d7 and (c) a real subject sample at peak concentration (Cmax) after oral administration of 50 mg atenolol + 12.5 mg chlorthalidone fixed dose formulation.
Biomed. Chromatogr. 2016; 30: 208–216
method employs deuterated ISs, while general ISs were used in these reports. Apart from one method in human breast milk (El-Gindy et al., 2008), all other methods relate to the analysis of these drugs in plasma samples. The developed UPLC-MS/MS method for ATN/CHL is at least 4/20 times more sensitive than reported methods. Further, the chromatographic analysis time is also the shortest as compared with available methods. A comparative overview of these methods and the present method are summarized in Table 1. Method validation There was minimal carry-over (≤0.11% of LLOQ area) during the autosampler carryover experiment in the extracted blank plasma, injected after the highest concentration of ATN (500 ng/mL) and CHL (150 ng/mL). The best fittings of the calibration curves were obtained by linear regression with 1/x2 weighting in the concentration range of 0.500–500 ng/mL (r2, 0.9999) for ATN and 0.250–150 ng/mL (r2, 0.9998) for CHL. The mean linear equations obtained were y = (0.004957 ± 0.000014) x + (0.000126 ± 0.000012) for ATN and y = (0.010980 ± 0.000083) x + (0.000143 ± 0.000094) for CHL. The accuracy and precision
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
211
reported for these drugs. Thus, protein precipitation, being the simplest and an efficient technique for gross removal of proteins was tested with common precipitants like methanol, ethanol, acetonitrile and perchloric acid. However, the recovery was not consistent at lower QC levels, especially for ATN. Similar results were obtained by liquid–liquid extraction with different organic diluents like methyl tert-butyl ether, dichloromethane-2-propanol (El-Glindy et al., 2008) and ethyl acetate. Although the recovery of the analytes was in the range of 60–75%, it was inconsistent. Thus, solid-phase extraction, which provides cleaner extracts through selective retention of the analytes on the column phase and for effective removal of endogenous moieties in plasma, was tested on a Phenomenex Strata-X (30 mg, 1 cm3) extraction cartridge. Washing with 1.0 mL water was adequate to remove any endogenous components and elution with 0.1% formic acid–acetonitrile (25:75, v/v) provided precise and quantitative recovery for both the analytes. The developed method was superior to the existing methods for their simultaneous determination in biological fluids with respect to sensitivity, throughput and sample processing volume (Elgawish et al., 2011; El-Glindy et al., 2008; Giachetti et al., 1997; Gonzalez et al., 2010; Khuroo et al., 2008). Additionally, the present
J. V. Shah et al.
Figure 2. (Continued)
212
(CV) observed for the calibration curve standards ranged from 97.7 to 102.3 and from 1.30 to 3.77% for ATN and from 97.6 to 102.5 and from 1.16 to 2.81% for CHL, respectively. The lowest concentration (LLOQ) in the standard curve that was measured with acceptable accuracy and precision was 0.50 and 0.250 ng/mL for ATN and CHL, respectively, at a signalto-noise ratio (S/N) of ≥10. The intra- and inter-batch precision and accuracy were established at five QC levels in validation runs performed at five QC levels (Table S1 in the Supporting Information). The intra-batch precision (CV) ranged from 2.37 to 5.73 and the accuracy was within 96.1–103.2% for both the analytes. Likewise, for the interbatch experiments, the precision varied from 2.72 to 5.91 and the accuracy was within 96.1–103.0%. The extraction recovery of analytes from solid-phase extraction ranged from 95.4 to 102.7 for ATN and from 95.6 to 103.2% for CHL. The mean recovery of ATN-d7 and CHL-d4 was 98.2 and 97.8%, respectively. The matrix effect is responsible for suppression or enhancement in the measurement of analyte signal owing to endogenous or exogenous components present in biological fluids. The absolute matrix effect, expressed as matrix factor (MF) was evaluated at four QC levels. The MFs were calculated from the peak area response for the analytes and their ISs
wileyonlinelibrary.com/journal/bmc
separately and their ratios were then used to find the IS-normalized MF, which ranged from 0.95 to 1.06 across four QC levels for both of the analytes (Table 2). Further, relative matrix effect was assessed in eight different plasma sources (five normal K3EDTA, one heparinized, one haemolysed and one lipemic). The precision (CV) in the measurement of slope of calibration lines was 2.68 and 2.02 for ATN and CHL respectively as shown in Table S2 in the Supporting Information. The samples kept for short-term and long term stock and working solutions of ATN, CHL and ISs remained unaffected up to 26 h and 25 days, respectively. The bench-top stability of analytes in plasma was determined up to 18 h and for five freeze–thaw cycles. Autosampler stability of the spiked QC samples maintained at 5 °C was established up to 48 h and at ambient temperature up to 12 h without significant loss of analytes. The dry extracts kept at 20 °C were stable up to 18 h. The samples stored for assessment of longterm stability of analytes in plasma were found stable for a minimum period of 70 days. The detailed stability results are summarized in Table 3. The precision (CV) and accuracy for method ruggedness with different columns and analysts ranged from 2.74 to 4.05% and 94.3 to 101.5%, respectively, across five QC levels for both of the analytes. While the precision (CV) for dilution reliability of 1/5th
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 208–216
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
LC-MS-MS; 10–2050 for ATN and 15–3035 for CHL
LC-MS-MS; 2.0–1000 for ATN and 5.0–500 for CHL
UPLC-MS-MS; 0.5–500 for ATN and 0.25–150 for CHL
4
5a
6
Supelcosil LC18 (250 × 4 mm, 5.0 μm); ATN – 15.70 min, CHL – 6.05 min; 22.0 min Luna CN (250 × 4.6 mm, 5.0 μm); ATN – 9.5 min, CHL – 5.0 min; 12.0 min Shim-pack® CN-propyl (250 × 4.6 mm, 5.0 μm); ATN – ~5.2 min, CHL – ~8.5 min; ~12.0 min Xterra C18 LC (150 × 4.6 mm, 5.0 μm); ATN – 3.54, CHL – 2.11; 3.0 min Luna C18 (150 × 4.6 mm, 3.0 μm); ATN – 4.5, CHL – 7.2; 1 2.0 min Acquity BEH C18 (50 × 2.1 mm, 1.7 μm); ATN – 0.97, CHL – 1.47; 2.0 min
LLE; 1000 μL plasma; salbutamol and xipamide
SPE; 100 μL human plasma; atenolol-d7 and chlorthalidone-d4
PP; 500 μL human plasma; pravastatin
SPE; 300 μL human plasma; metoprolol and hydrochlorothiazide
PP; 960 μL human plasma; hydrochlorothiazide
LLE; 1000 μL breast milk; guaifenesin
Column; retention time; run time
Extraction; sample volume; internal standard
Bioequivalence study with 100 + 25 mg of ATN + CHL tablets in 23 healthy subjects Measurement of plasma concentration of ATN, CHL and other cardiovascular drugs in 14 patients Bioequivalence study with 50 + 12.5 mg of ATN + CHL tablets in 28 healthy subjects
Determination of ATN and CHL in spiked human plasma
Pharmacokinetic studies with 100 + 25 mg and 50 + 12.5 mg of ATN + CHL tablets in 18 healthy subjects Determination of atenolol and chlorthalidone in human breast milk
Application
PM
Gonzalez et al. (2010)
Khuroo et al. (2008)
Elgawish et al. (2011)
El-Gindy et al. (2008)
Giachetti et al. (1997)
Reference
a Together with other cardiovascular drugs. ATN, Atenolol; CHL, chlorthalidone; LLE, liquid–liquid extraction; SPE, solid-phase extraction; PP, protein precipitation extraction, PM, present method.
HPLC-UV; 100–10,000 for ATN and CHL
3
HPLC-UV for CHL and Fluorescence for ATN; 10–1000 for ATN and CHL
Detection technique; linearity (ng/mL)
HPLC-UV; 300–20,000 for ATN and 250–5000 for CHL
213
Biomed. Chromatogr. 2016; 30: 208–216
2
1
Serial No.
Table 1. Comparison of salient features of chromatographic methods developed for simultaneous determination of atenolol and chlorthalidone
Determination of atenolol and chlorthalidone in plasma
J. V. Shah et al. Table 2. Extraction recovery and matrix factor for atenolol and chlorthalidone QC level
Area response (replicate, n = 6) A (post-extraction spiking)
Atenolol LQC MQC-2 MQC-1 HQC Chlorthalidone LQC MQC-2 MQC-1 HQC
B (pre-extraction spiking)
C (neat samples in mobile phase)
Extraction recovery, % (B/A)
Matrix factor Analyte (A/C)
IS
ISnormalized
2,357 39,171 313,335 626,652
2,248 38,456 321,795 602,839
2,507 38,402 319,729 646,032
95.4 (95.8)a 98.2 (97.3)a 102.7 (103.1)a 96.2 (96.4)a
0.94 1.02 0.98 0.97
0.99 0.96 1.03 0.98
0.95 1.06 0.95 0.99
1,163 23,265 139,554 186,135
1,200 22,427 133,413 182,040
1,129 23,034 145,368 195,931
103.2 (102.4)b 96.4 (96.7)b 95.6 (95.1)b 97.8 (97.2)b
1.03 1.01 0.96 0.95
1.01 0.95 0.99 0.97
1.02 1.06 0.97 0.98
a
Values for atenolol-d7. values for chlorthalidone-d4. IS, Internal standard; LQC: low quality control; MQC: medium quality control; HQC: high quality control.
b
Table 3. Stability of atenolol and chlorthalidone in plasma under various conditions (replicates, n = 6) Storage conditions
Bench-top stability at room temperature, 18 h Freeze–thaw stability at 20 °C Freeze–thaw stability at 70 °C Processed sample stability room temperature, 12 h Autosampler stability at 5 °C, 48 h Dry extract stability at 5 °C, 18 h Long-term stability at 20 °C, 70 days Long-term stability at 70 °C, 70 days
Atenolol
Chlorthalidone
Nominal concentration (ng/mL)
Mean stability sample (ng/mL) ± SD
Percentage change
Nominal concentration (ng/mL)
Mean stability sample (ng/mL) ± SD
Percentage change
400.0 1.500 400.0 1.500 400.0 1.500 400.0 1.500 400.0 1.500 400.0 1.500 400.0 1.500 400.0 1.500
412.7 ± 3.32 1.561 ± 0.027 383.2 ± 5.77 1.445 ± 0.028 411.8 ± 7.47 1.578 ± 0.008 386.2 ± 4.34 1.570 ± 0.023 392.2 ± 5.22 1.592 ± 0.027 406.0 ± 4.31 1.420 ± 0.031 390.2 ± 3.80 1.409 ± 0.024 416.4 ± 4.26 1.435 ± 0.020
3.18 4.06 4.20 3.66 2.95 5.20 3.45 4.66 1.95 6.13 1.50 5.33 2.45 6.07 4.10 4.33
120.0 0.750 120.0 0.750 120.0 0.750 120.0 0.750 120.0 0.750 120.0 0.750 120.0 0.750 120.0 0.750
121.9 ± 1.26 0.707 ± 0.007 124.5 ± 1.60 0.726 ± 0.006 117.9 ± 1.96 0.782 ± 0.008 116.0 ± 2.21 0.795 ± 0.005 122.8 ± 2.30 0.773 ± 0.005 115.0 ± 1.39 0.793 ± 0.004 124.9 ± 1.04 0.717 ± 0.008 116.5 ± 1.64 0.706 ± 0.008
1.58 5.73 3.75 3.20 1.75 4.27 3.33 6.00 2.33 3.06 4.17 5.73 4.08 4.40 2.92 5.87
SD, Standard deviation; n, number of replicates. Mean stability samples – Mean comparison samples Percentage change ¼ 100 Mean comparison samples
and 1/10th dilution were between 0.96 and 3.75%, and the accuracy ranged from 96.2 to 102.9%.
214
Application of the method The aim of the study was to test the comparative bioavailability of a test and reference product under fasting and thereby to determine their bioequivalence. To minimize variability and focus
wileyonlinelibrary.com/journal/bmc
on the comparison of the two formulations, healthy volunteers were selected in the present work. The validated method was applied using a low-dose combination of ATN (50 mg) and CHL (12.5 mg) in healthy subjects for better efficacy-to-side effect ratio as reported previously (Carter et al., 2004; Pareek et al., 2008). Figure 3 shows the time course profile of ATN and CHL plasma concentration under fasting. The mean pharmacokinetic parameters evaluated after oral administration of combination tablet
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 208–216
Determination of atenolol and chlorthalidone in plasma Table 4. Mean pharmacokinetic parameters following oral administration of 50 mg atenolol + 12.5 mg chlorthalidone combination formulation in 28 healthy Indian subjects under fasting Parameter
Cmax (ng/mL) Tmax (h) t1/2 (h) AUC 0–120 (h ng/mL) AUC 0–inf (h ng/mL) Kel (1/h)
Atenolol (mean ± SD)
Chlorthalidone (mean ± SD)
Test
Reference
Test
Reference
321.6 ± 7.9 3.11 ± 0.28 7.75 ± 0.22 2387.7 ± 32.6 2507.1 ± 38.3 0.089 ± 0.018
315.2 ± 6.6 3.07 ± 0.20 7.51 ± 0.14 2316.5 ± 29.8 2432.3 ± 37.2 0.092 ± 0.014
74.8 ± 3.6 5.97 ± 0.19 31.7 ± 0.15 1048.4 ± 20.3 1093.5 ± 26.1 0.022 ± 0.003
73.3 ± 3.9 5.74 ± 0.13 31.5 ± 0.10 1019.6 ± 23.6 1066.5 ± 25.2 0.023 ± 0.003
Cmax, Maximum plasma concentration; Tmax, time point of maximum plasma concentration; t1/2, half life of drug elimination during the terminal phase; AUC0–t, area under the plasma concentration-time curve from 0 to 120 h; AUC0–inf, area under the plasma concentration-time curve from zero hour to infinity; Kel, elimination rate constant; SD, standard deviation. Reference formulation: Tenoretic®, 50 mg atenolol and 12.5 mg chlorthalidone tablets from AstraZeneca UK Limited, Luton, UK. Test formulation: 50 mg atenolol and 12.5 mg chlorthalidone tablets from a Generic Company, India.
115 study samples were selected which were near the Cmax and the elimination phase in the pharmacokinetic profile of the drugs. These samples were reanalyzed and the results were compared with the initial study results. The percentage change in the results was within ±14% for both the analytes, which is within the acceptance criterion. The graphical representation of the results is shown in Figure S2 in the Supporting Information.
Conclusion A highly sensitive, rapid and rugged UPLC-MS/MS method has been developed for the simultaneous quantitation of ATN and CHL in human plasma. Absence of matrix interference was suitably verified through IS-normalized matrix factors and by calculation of precision in the measurement of slope of calibration curves prepared in different plasma sources. The statistical treatment data reveal high accuracy and precision of the proposed method for all of the validation parameters. The method was successfully applied to measure plasma concentration of ATN and CHL in subject samples. Finally, the results of incurred sample reanalysis confirm the reproducibility of the proposed method.
Acknowledgments Figure 3. Mean plasma concentration–time profile of atenolol and chlorthalidone after oral administration of test (50 mg atenolol + 12.5 mg chlorthalidone tablets from a Generic Company) and a reference (TENORETIC®, 50 mg atenolol + 12.5 mg chlorthalidone tablets from AstraZeneca UK Limited, Luton, UK) formulation to 28 healthy subjects.
One of the authors, Jaivik V. Shah wishes to thank UGC, New Delhi for BSR Fellowship and to the management of Cadila Pharmaceuticals Ltd, Ahmedabad, India for providing instrumentation and infrastructure facility to carry out this work.
References
Biomed. Chromatogr. 2016; 30: 208–216
Arias R, Jimenez RM, Alomso RM, Telez M, Arrieta I, Flores P and Lastra EO. Determination of the β-blocker atenolol in plasma by capillary zone electrophoresis. Journal of Chromatography A 2001; 916: 297–304. Belal F, El-Brashy AM, El-Enany N and Tolba MM. Simultaneous determination of celiprolol HCl and chlorthalidone in tablets and biological fluids using high-performance liquid chromatography. Acta Chromatographica 2012; 24: 185–206. Bhatia NM, Gurav SB, Jadhav SD and Bhatia MS. RP-HPLC method for simultaneous estimation of atorvastatin calcium, losartan potassium, atenolol and aspirin from tablet dosage form and plasma. Journal of Liquid Chromatography and Related Technologies 2012; 35: 428–443.
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
215
are summarized in Table 4. The mean peak concentration (Cmax), time to reach Cmax (Tmax) and area under the concentration–time curve (AUC0–120 h) were 321.6 ± 7.9 ng/mL, 3.11 h and 2387.7 h. ng/mL for ATN and 74.8 ng/mL, 5.97 h and 1048.4 h. ng/mL for CHL, respectively. These values were in agreement with a previous method with healthy Indian subjects based on dose strength (Khuroo et al., 2008). Further, CHL had a long half-life of 31.7 h compared with 7.75 h for ATN as described in several reports (TENORETIC®, ). To prove the method’s reproducibility,
J. V. Shah et al. Braza AJ, Modamio P and Marino EL. Two reproducible and sensitive liquid chromatographic methods to quantify atenolol and propranolol in human plasma and determination of their associated analytical error functions. Journal of Chromatography B 2000; 738: 225–231. Carter BL, Ernst ME and Cohen JD. Hydrochlorothiazide versus chlorthalidone: evidence supporting their interchangeability. Hypertension 2004; 43: 4–9. Chiap P, Buraglia M, Ceccato A, Hubert P and Crommen J. Automated liquid chromatographic determination of atenolol in plasma using dialysis and trace enrichment on a cation-exchange precolumn for sample handling. Journal of Chromatography B 2000; 739: 205–217. Damiani PC. Determination of atenolol in human urine by emissionexcitation fluorescence matrices and unfolded partial least-squares with residual bilinearization. Talanta 2011; 85: 1526–1534. D’Orazio G and Fanali S. Use of tecicoplanin stationary phase for the enantiomeric resolution of atenolol in human urine by nano-liquid chromatography–mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2006; 40: 539–544. Elgawish MS, Mostafa SM and Elshanawane AA. Simple and rapid HPLC method for simultaneous determination of atenolol and chlorthalidone in spiked human plasma. Saudi Pharmaceutical Journal 2011; 19: 43–49. El-Glindy A, Sallam S and Abdel-Salam RA. HPLC method for the simultaneous determination of atenolol and chlorthalidone in human breat milk. Journal of Separation Science 2008; 31: 677–682. Feleuren HLJM and Rossum JMV. Determination of chlorthalidone in plasma, urine and red blood cells by gas chromatography with nitrogen detection. Journal of Chromatography 1978; 152: 41–54. Giachetti C, Tenconi A, Canali S and Zanolo G. Simultaneous determination of atenolol and chlorthalidone in plasma by high-performance liquid chromatography. Application to pharmacokinetic studies in man. Journal of Chromatography B 1997; 698: 187–194. Gonzalez O, Iriarte G, Rico E, Ferreiros N, Maguregui MI, Alonso RM and Jimenez RM. LC-MS/MS method for the determination of several drugs used in combined cardiovascular therapy in human plasma. Journal of Chromatography B 2010; 878: 2685–2692. Guelen PJM, Baars AM and Vree TB. Rapid and sensitive determination of chlorthalidone in blood, plasma and urine of man using highperformance liquid chromatography. Journal of Chromatography 1980; 181: 497–503. Iha MH, Martinez AS and Bonato PS. Enantioselective analysis of atenolol in biologic fluids: comparison of liquid–liquid and solid-phase extraction methods. Journal of Chromatography B 2002; 767: 1–9. Johnson RD and Lewis RJ. Quantification of atenolol, metraprolol, and propanolol in postmortem human fluid and tissue specimens via LC/APCI-MS. Forensic Science International 2006; 156: 106–117. Khuroo A, Mishra S, Singh O, Saxena S and Monif T. Simultaneous determination of atenolol and chlorthalidone by LC-MS-MS in human plasma. Chromatographia 2008; 68: 721–729. Lawson G, Cocks E and Tanna S. Quantitative determination of atenolol in dried blood spot samples by LC-HRMS: a potential method for assessing medication adherance. Journal of Chromatography B 2012; 897: 72–79. Leite FS, Pereira VA, Omosako CE, Carmona MJC, Auler JOC and Santos SRCJ. A micromethod for the quantification of atenolol in plasma using high-performance liquid chromatography with fluorescence detection. Therapeutic Drug Monitoring 2006; 28: 237–244.
Li W, Li Y, Francisco DT and Naidong W. Hydrophilic interaction liquid chromatographic tandem mass spectrometric determination of atenolol in human plasma. Biomedical Chromatography 2005; 19: 385–393. Muirhead DC and Christie RB. Simple, sensitive and selective highperformance liquid chromatographic method for analysis of chlorthaidone in whole blood. Journal of Chromatography 1987; 416: 420–425. Pareek A, Karnik N, Salagre SB, Zawar SD, Joglekar VK, Chandurkar N and Naik GS. Clinical effectiveness of low-dose chlorthalidone (6.25 mg) + atenolol combination in stage I hypertensive patients: a multicenter, randomized, controlled study. Current Medical Research and Opinion 2008; 24: 1771–1779. Rosenberg MJ, Lam KK and Dorsey TE. Analysis of chlorthaidone in whole blodd by high-performance liquid chromatography. Journal of Chromatography 1986; 375: 438–443. Salado SC and Vera-Avila LE. On-line solid-phase extraction and highperformance liquid chromatographic determination of chlorthalidone in urine. Journal of Chromatography B 1997; 690: 195–202. Sharma P, Patel DP, Sanyal M, Berawala H, Guttikar S and Shrivastav PS. Simultaneous analysis of oxybutynin and its active metabolite N-desethyl oxybutynin in human plasma by stable isotope dilution LC-MS/MS to support a bioequivalence study. Journal of Pharmaceutical and Biomedical Analysis 2013; 84: 244–255. Shrivastav PS, Buha SM and Sanyal M. Detection and quantitation of betablockers in plasma and urine. Bioanalysis 2010; 2: 263–276. Spanakis M and Niopas I. Determination of atenolol in human plasma by HPLC with fluorescence detection: validation and application in a pharmacokinetic study. Journal of Chromatographic Science 2013; 52: 128–132. Sweetman SC. , 33rd edn, Martindale – The Complete Drug Reference. Pharmaceutical Press, Royal Pharmaceutical Society of Great Britain: London , 2002. TENORETIC® (atenolol and chlorthalidone). Product details. AstraZeneca Pharmaceuticals, London, n.d. Available from: http://www1. astrazeneca-us.com/pi/tenoretic_lpv.pdf (accessed December 2014). US Food and Drug Administration. Guidance for Industry: ICH E6 Good Clinical Practice. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research and Centre for Biologics Evaluation and Research, 1996. US Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation. U.S. Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research and Centre for Veterinary Medicine, 2001. Yadav M and Shrivastav PS. Incurred sample reanalysis: a decisive tool in bioanalytical research. Bioanalysis 2011; 3: 1007–1024. Yilmaz B, Arslan S and Asci A. HPLC method for determination of atenolol in human plasma and application to a pharmacokinetic study in Turkey. Journal of Chromatographic Science 2012; 50: 914–919.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
216 wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 208–216
