The drug standards for atenolol, nadolol, bisoprolol, carvedilol, acebutolol, labetalol, propranolol hydrochloride, and metoprolol tartrate were purchased from Sigma-Aldrich (Tokyo, Japan). Timolol maleate, arotinolol, and betaxolol hydrochloride were purchased from the Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Carteolol hydrochloride, esmolol hydrochloride, celiprolol hydrochloride, alprenolol hydrochloride, bevantolol hydrochloride, and atenolol-d7 (internal standard: IS) were purchased from Toronto Research Chemicals (Ontario, Canada). Pindolol, acetonitrile (LC-MS grade, >99.9% purity), methanol (LC-MS grade, 99.7% purity), ultrapure water (LC-MS grade), acetic acid (analytical grade), and ammonium acetate (analytical grade) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Drug standards for α-hydroxy metoprolol, O-desmethyl metoprolol, and rac metoprolol-d7 (internal standard: IS) were purchased from Toronto Research Chemicals (Ontario, Canada). 4-hydroxy propranolol hydrochloride was purchased from Cayman Chemical (Wisconsin, USA). Pooled normal human serum and urine samples were purchased from Millipore (Billerica, MA, USA) [1][2]. The Oasis^® PRiME HLB extraction cartridges were purchased from Waters (Milford, MA, USA). All other chemicals were commercially available and were of reagent grade [1][2].

2.2.1. Preparation of Standard Solutions and Calibration Standards

(Method-A): Individual stock solutions (1 mg/mL concentration) of the compounds to be analyzed were prepared with methanol (stable for at least three months when stored at −20˚C). Standard serum solutions with concentrations of 20, 100, 200, and 400 ng/mL of each compound were prepared by diluting appropriate aliquots of the stock solution with drug-free serum (normal human serum). Human serum (0.5 mL) was added to water (0.5 mL) containing 10, 50, 100, or 200 ng of each of the 17 beta-blockers and 100 ng of IS. Calibration curves were obtained using simple linear regression analysis of the concentration of each drug and the corresponding peak area ratio. The regression equations for the 17 beta-blockers extracted from the human serum were based on the ratio of the peak area of each drug to that of the IS [1][2].

(Method-B): Serum or urine standards were prepared with concentrations of 2, 10, 100, and 200 ng/mL of each compound (metoprolol, propranolol, and α-hydroxy metoprolol) and 10, 50, 100, and 200 ng/mL for 4-hydroxy propranolol by diluting appropriate aliquots of the stock solution with drug-free serum (normal human serum) or urine (normal human urine). In our study, 0.5 mL of human serum or urine was added to 0.5 mL of water containing 1, 5, 50, or 100 ng of metoprolol, propranolol, α-hydroxy metoprolol, O-desmethyl metoprolol, 5, 25, 50, or 100 ng of 4-hydroxy propranolol, and 50 ng metoprolol-d7 (IS). The calibration curves were prepared in the same manner as Method-A.

2.2.2. Apparatus and Chromatographic Conditions

(Method-A): LC-MS/MS experiments were performed using an HPLC system equipped with Shimadzu LC-20AD pumps (Shimadzu, Kyoto, Japan), a SIL-20AC autosampler (Columbia, MD, USA), and a 4000 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA).

Chromatographic separation was performed using a Mightysil-RP-18 MS column (2.0 mm × 150 mm, particle size 5 µm; Kanto Chemical Co., Inc., Tokyo, Japan). Two solvents were used for gradient elution: a) 10 mM ammonium acetate buffer and b) acetonitrile. The mobile phase composition was initially held at 90% (A) for 0.1 min and then changed during linear gradient elution from 90% (A) to 40% (A) over 10 min. The mobile phase was returned to 90% (A) for 5 min before the next run. The flow rate was set to 0.20 mL/min and 10 µL of sample was injected for each analysis. The column and autosampler were maintained at 37˚C and 4˚C, respectively.

All experiments were conducted in the positive-ion electrospray mode. The TurboIonSpray source was operated at 600˚C with a capillary voltage of 5500 V. Nitrogen was used as the nebulizer, curtain (40 psi), and collision (4 psi) gases. The optimal chromatographic and mass spectrometric conditions for the analysis of all compounds were determined by injecting pure standard solutions into the LC-MS/MS system. Simultaneous analysis was performed in the dMRM mode for 1 min before and after the retention time of each compound [1][2][10]-[12].

(Method-B): An Agilent 1290 liquid chromatograph, equipped with a Mightysil-RP-18 MS column (2.0 mm × 150 mm, particle size 5 µm; Kanto Chemical Co., Inc., Tokyo, Japan), in combination with a 6460 triple quadrupole mass spectrometer with a Jet Stream ESI source (Agilent Technologies, Santa Clara, CA, USA), was used for the analysis.

Mobile phase A consisted of 10 mM ammonium acetate and 0.1% acetic acid in ultrapure water, and mobile phase B was acetonitrile. The gradient elution program was as follows: the mobile phase composition was held initially at 90% (A) for 0.1 min and then changed during the linear gradient elution from 90% (A) to 40% (A) over 10 min. The mobile phase was then returned to 100% (A) for 5 min for the next analysis. The flow rate was 0.2 mL/min. The autosampler was maintained at 4˚C. The column oven temperature was 37˚C. The ESI parameters were capillary voltage, 4000 V; nebulizer gas (N2) pressure, 50 psi; drying gas (N2) flow rate, 10 L/min at 300˚C; sheath gas (N2) flow rate, 12 L/min at 350˚C. The optimal chromatographic and mass spectrometric conditions for the analysis of all the compounds were obtained by injecting pure standard solutions into the LC/MS/MS system. All experiments were measured in product ion scan mode to confirm the retention time. Simultaneous analysis was performed in dMRM mode for 1 min before and after the retention time of each compound. The determination of the MS/MS parameters and data acquisition was performed using MassHunter Workstation Software (version B.07.00, Agilent Technologies).

2.2.3. Extraction

(Method-A): The drugs were extracted from human serum using an Oasis^® PRiME HLB cartridge column. These samples were analyzed to determine possible endogenous interferences and were used as “blanks.” An Oasis^® PRiME HLB cartridge column with a 1 mL capacity was placed in an Agilent Vac Elut system (Agilent Technologies).

Human serum (0.5 mL) was added to water (0.5 mL) containing 10 - 200 ng of each of the 17 beta-blockers and 100 ng of IS. The mixture was loaded onto an Oasis^® PRiME HLB cartridge column and allowed to pass through it. The column was then washed with 1 mL of a 5% aqueous methanol solution. Beta-blockers were eluted with 1 mL of 90% methanol and 10% acetonitrile. The eluent was then dried under N2 gas at 40˚C. The residue was dissolved in 200 µL of the initial mobile phase composition (A:B = 90:10), and an aliquot (10 µL) was analyzed using a chromatographic system (Figure 3) [1][2][10]-[12].

(Method-B): 0.5 mL of human serum or urine was added to 0.5 mL of water containing 1 - 200 ng of each of the beta blockers, their metabolites, and 50 ng metoprolol-d7 (IS). The extraction methods were performed in the same manner as Method-A, and an aliquot (1 µL) was then analyzed by the chromatographic system (Figure 3).

Figure 3. Extraction method of beta-blockers and their metabolites using an Oasis^® PRiME HLB column.

2.2.4. Linearity, Accuracy, Precision, and Recovery

(Method-A): Analyses of the 17 beta-blockers in serum samples were performed on three separate days using freshly prepared samples and solutions to evaluate the linearity of the standard calibration curve. Calibration curves were prepared over a linear range of 20 - 400 ng/mL using four serum concentrations (20, 100, 200, and 400 ng/mL). Linearity was evaluated by analyzing six replicates.

Intraday assay precision and accuracy were obtained by analyzing six aliquots of quality control samples in duplicate using a calibration curve constructed on the same day. Interday assay precision and accuracy were evaluated using six replicate determinations for each concentration with solutions prepared on different days. Matrix effects and extraction recovery were verified. The matrix effect and extraction recovery were estimated at four analyte concentrations (20, 100, 200, and 400 ng/mL for beta-blockers, n = 6) in the serum. The recoveries were calculated by comparing the chromatographic peak areas obtained from the extracts of the serum samples (containing water) with those obtained by adding beta-blockers to the extracted solution of blank serum [1]-[12].

(Method-B): The calibration curves were prepared over a linear range of 2 - 200 ng/mL using four concentrations (2, 10, 100, and 200 ng/mL for metoprolol, propranolol, α-hydroxy metoprolol, and O-desmethyl metoprolol; 10, 50, 100, and 200 ng/mL for 4-hydroxy propranolol) in serum or urine.

The intraday assay precision, accuracy, the matrix effect, and extraction recovery were performed in the same manner as Method-A. The matrix effect and extraction recovery were estimated at three analyte concentrations (10, 100, and 200 ng/mL, n = 5) for the beta blockers and their metabolites in serum or urine. The recoveries were calculated as in Method-A.

2.2.5. Stability

The stability of the 17 beta-blockers (Method-A), 2 major metabolites (Method-B), and IS in serum and urine was investigated at various concentrations. Freshly prepared samples were evaluated at room temperature, 4˚C, and −20˚C over 12 days. Freeze/thaw stability was determined after three freeze/thaw cycles. Stability tests were performed by analyzing two replicates for each sample [1][2][10]-[12].

2.2.6. Limits of Detection and Quantification (LOD and LOQ)

The LOD was determined as the injection volume required to produce a peak with a signal-to-noise ratio of at least 3:1. The LOQ was defined as the lowest concentration on the standard curve that could be measured with acceptable accuracy (coefficient of variation, CV < 10.0%). The LOQ for the 17 beta-blockers in serum, as well as the beta-blockers and their metabolites in serum or urine, were determined using LC-MS/MS with ESI after solid-phase extraction (Oasis^® PRiME HLB cartridge column) [1][2][10]-[12].

In Method A, a 4000 QTRAP mass spectrometer (Applied Biosystems) system was used to analyze 17 beta-blockers, and in Method B, a 6460 triple quadrupole mass spectrometer (Agilent Technologies) was used to analyze two types of drugs and their major metabolites.

(Method-A): The positive-ion electrospray mode was selected, and the collision energies were optimized for each drug. All the precursor ions (Q1) of the 17 beta-blockers were (M + H)⁺. The (M + H)⁺ ion peak was clearly detected, and the product ions were obtained from the mass spectrum of each drug with sufficient detection sensitivity (Table 1). Good separation of the 17 beta-blockers is observed using LC-MS/MS, as shown in the chromatograms (Figure 3). The positive-ion electrospray mode is selected, and the collision energies are optimized for each drug (Figure 2). All the precursor ions (Q1) of the 17 beta-blockers were (M + H)⁺. The (M + H)⁺ ion peak is clearly detected and the product ions are obtained from the mass spectrum of each drug with sufficient detection sensitivity (Table 1, Figure 4) [1][2].

(Method-B): The positive ion electrospray mode was selected, and optimal collision energies were optimized for each drug. All precursor ions (Q1) of metoprolol, propranolol, and their metabolites (α-hydroxy metoprolol, O-desmethyl metoprolol, and 4-hydroxy propranolol) showed the [M + H]⁺. The [M + H]⁺ ion peak was detected clearly, and product ions were obtained in the mass spectrum of each drug, with sufficient detection sensitivity (Table 2). In addition, the separation of metoprolol, propranolol, α-hydroxy metoprolol, O-desmethyl metoprolol, and 4-hydroxy propranolol by LC-MS/MS was good, as seen on the dMRM chromatograms (Table 2, Figure 5).

Table 1. Retention time and tandem mass spectrometry parameters of beta-blockers.

1. Atenolol-d7 (IS), 2. Atenolol, 3. Carteolol, 4. Nadolol, 5. Pindolol, 6. Timolol, 7. Acebutolol, 8. Arotinolol, 9. Metoprolol, 10. Esmolol, 11. Celiprolol, 12. Labetalol, 13. Bisoprolol, 14. Propranolol, 15. Alprenolol, 16. Betaxolol, 17. Bevantolol, 18. Carvedilol. Injection volume: 5 ng of each beta blocker and Atenolol-d7 (IS).

Figure 4. TIC and tandem mass chromatograms of beta-blockers and Atenolol-d7 (IS) extracted from human serum.

Table 2. Retention time and tandem mass spectrometry parameters of metoprolol, propranolol, and their metabolites.

Figure 5. Dynamic multiple reaction monitoring chromatograms of metoprolol, propranolol, their metabolites, and metoprolol-d7 (IS) extracted from serum. Column: Mightysil-RP-18 MS column (2.0 mm × 150 mm, particle size 5 µm). Injection volume: 0.25 ng of each drug.

(Method-A): The calibration curve (ratio of the peak area to the concentration of each drug) was linear over the entire concentration range of 20 - 400 ng/ml. The coefficients of determination (r) from the regression analyses of the drugs are in the range of 0.9686 - 0.9997 (Table 3). In our LC-MS/MS with ESI method for the simultaneous determination of 17 beta-blockers, the precision was satisfactory, with CV values always lower than 10.0% for intraday, interday, and total assay reproducibility. The recoveries of the 17 beta-blocker drugs were in the range of 66.1% - 93.5%, and a precision within 7.0% CV was acceptable for this method. However, the extraction yields of bevantolol and carvedilol were low (37.5% and 26.2%, respectively) (Table 4). No significant interference was observed at the retention times of the analytes and the IS, indicating acceptable selectivity. However, the matrix effect is insignificant (Figure 6) [1][2]. The primary objective of this study is to conduct a broad-scale screening of low-concentration beta-blockers. While the extraction recovery rate may be low for some compounds, the overall sensitivity, linearity, and ability of this method to detect these compounds within the acceptable error range make it suitable for initial-stage screening.

Table 3. Retention time and tandem mass spectrometry parameters of metoprolol, propranolol, and their metabolites.

Table 4. Extraction yields, limits of detection, and limits of quantification of beta-blockers using LC-MS/MS.

Figure 6. Tandem mass chromatograms of Atenolol-d₇ (IS) extracted from human serum. Injection volume: 5 ng.

(Method-B): The calibration curve (the ratio of the peak area to the concentration of each drug) was linear over the concentration range of 2 - 200 ng/mL (metoprolol, propranolol, α-hydroxy metoprolol, and O-desmethyl metoprolol). The calibration curve of 4-hydroxy propranolol was linear over the concentration range of 10 - 200 ng/mL. The coefficients of determination (r) from the regression analyses of the drugs were between 0.9246 and 0.9997 (n = 5: Table 5).

Table 5. Calibration curves of metoprolol, propranolol, and their metabolites.

In our LC-MS/MS with ESI method for simultaneous determination of the beta blockers and their metabolites, the precision was satisfactory, with CV values always lower than 10.0% for the intraday, interday, and total assay reproducibility. The recoveries at 50 ng of metoprolol, propranolol, α-hydroxy metoprolol, O-desmethyl metoprolol, and 4-hydroxy propranolol in serum ranged from 96.8 to 106.5%, with precision within 5.9% CV values; in urine, the recoveries ranged from 93.8% to 99.0%, with precision within 4.1% CV values, which were acceptable for this method. However, the extraction yields of 4-hydroxy propranolol were low (in serum 54.4%, in urine 54.1%). Currently, we are experimenting to improve the extraction yields of 4-hydroxy propranolol (Table 6, Table 7). No significant interferences were observed at the retention times of the analytes and IS, indicating acceptable method selectivity. The matrix effect was insignificant.

Table 6. Extraction yields, limits of detection, and limits of quantification of metoprolol, propranolol, and their metabolites from serum using LC-MS/MS.

Table 7. Extraction yields, limits of detection, and limits of quantification of metoprolol, propranolol, and their metabolites from urine using LC-MS/MS.

(Method-A): A stability study was conducted to determine the optimal storage temperature for serum samples. The results indicated that the 17 beta-blockers and IS were stable for up to 24 h at room temperature. Furthermore, these drugs were stable for 10 days at 4˚C and −20˚C and for at least two weeks. Therefore, all extracted samples were refrigerated at 4˚C for same-day analysis, whereas serum samples were frozen at −20˚C or −80˚C until LC-MS/MS analysis [1][2].

(Method-B): A stability study was conducted to determine the best storage temperature for the serum or urine samples. The results indicated that metoprolol, propranolol, α-hydroxy metoprolol, O-desmethyl metoprolol, 4-hydroxy propranolol, and the IS were stable up to 12 h at room temperature. Furthermore, these drugs were stable up to 12 days when stored at 4˚C and −20˚C. Therefore, all extracted samples were stored refrigerated at 4˚C for same-day analysis, whereas serum and urine samples were frozen at −20˚C until their analysis by LC-MS/MS.

(Method-A): The LODs of atenolol, carteolol, nadolol, pindolol, timolol, acebutolol, arotinolol, metoprolol, esmolol, celiprolol, labetalol, bisoprolol, propranolol, alprenolol, betaxolol, bevantolol, and carvedilol were 0.048, 0.016, 0.044, 0.0034, 0.14, 0.040, 0.040, 0.017, 1.6, 0.013, 0.11, 0.026, 0.036, 0.033, 0.22, 0.03, and 0.040 ng/mL, respectively, and the LOQs of the 17 beta-blockers were 20.0 ng/mL (Table 4) [1][2].

These results suggest that the detection sensitivity of this analytical method is acceptable for measuring the therapeutic concentrations [33] of these drugs. Therefore, our analytical method can be used to determine the therapeutic range of beta-blocker concentrations.

For the GC-MS analysis, it is necessary to gasify the sample, and the analyzable samples are limited to relatively low-molecular-weight gases, volatile compounds, or thermally stable compounds. In LC-MS/MS, it is possible to analyze volatile and thermally unstable compounds as long as they are dissolved in the mobile phase, which has the advantage that the application range of the analytical samples is wider than that of GC-MS. Brunetto M.d.R., et al. [19] developed a method for the determination of atenolol and propranolol in human plasma samples by GC-MS. The LOQ was approximately 6.0 ng/mL. Phyo Lwin E.M., et al. [25] developed a method for determining atenolol levels in human plasma and milk samples using LC-MS/MS. The LOD and LOQ values were in the range of 1.0 - 5.0 ng/mL for atenolol. Elmongy H. et al., [27] developed a method for determining metoprolol levels in human plasma using LC-MS/MS. The LOQ was approximately 1.5 ng/mL. Our LC-MS/MS with ESI method could measure compounds at approximately the same concentrations as the analytical methods of Brunetto M.d.R. et al., Phyo Lwin et al., and Elmongy et al. In our experiments, the LOD values were in the range of 0.0034 - 1.6 ng/mL, and the LOQ values were 20 ng/mL for the 17 beta-blockers, respectively. The matrix effect on the LOQ values is insignificant, and both accuracy and precision were sufficient. Therefore, our analytical method can measure beta-blockers in a manner similar to the methods mentioned above.

(Method-B): The LODs of metoprolol, propranolol, α-hydroxy metoprolol, O-desmethyl metoprolol, and 4-hydroxy propranolol were 0.50, 0.68, 0.69, 1.28, and 0.54 ng/mL, respectively, in the serum (Table 6). The LODs of metoprolol, propranolol, α-hydroxy metoprolol, O-desmethyl metoprolol, and 4-hydroxy propranolol were 0.65, 0.17, 1.0, 0.09, and 0.71 ng/mL, respectively, in the urine (Table 7). The LOQs of metoprolol, propranolol, α-hydroxy metoprolol, and O-desmethyl metoprolol were 2.0 ng/mL. The LOQ of 4-hydroxy propranolol was 10 ng/mL (Table 6, Table 7).

These results suggest that the detection sensitivity of this method is acceptable for the determination of therapeutic concentrations [33] of these drugs (for example, the therapeutic concentration of propranolol is 20 - 100 ng/mL).

In GC-MS analysis, it is necessary to gasify the sample, and the analyzable samples are limited to relatively low molecular gases, volatile compounds, or thermally stable compounds. In LC-MS/MS, it is possible to analyze compounds that are difficult to volatilize and thermally unstable compounds as long as they are dissolved in the mobile phase, which has the advantage that the application range of analytical samples is wider than that of GC-MS. Brunetto M.d.R., et al. [19] developed a method for the determination of atenolol and propranolol in human plasma samples by GC-MS. The LOQ was approximately 6.0 ng/mL. Elmongy H, et al. [27] developed a method for the determination of metoprolol in human plasma by LC-MS/MS. The LOQ was approximately 1.5 ng/mL. Our LC-MS/MS with ESI method was able to measure compounds at approximately the same concentrations as the analytical methods of Brunetto M.d.R., et al., Phyo Lwin E.M., et al., and Elmongy H, et al. In our experiments, the LOD and LOQ were in the range of 0.09 - 1.28 ng/mL and were 2.0 ng/mL for metoprolol, propranolol, α-hydroxy metoprolol, O-desmethyl metoprolol, and 4-hydroxy propranolol (the LOQ of 4-hydroxy propranolol was 10 ng/mL).