4.74.7
© Springer-Verlag Berlin Heidelberg 2005
II.4.7 β-Blockers
by Makoto Ueki
Introduction
β-Blockers ( β-adrenergic receptor antagonists) block the e ects of catecholamines on signal
transmission through β-receptors; they cause hypotension by decreasing the heart beat rate
and cardiac output, and prevent the attacks of arrhythmia and angina pectoris. In the brain, the
drugs attenuate migraine by suppressing the dilation of blood vessels; in the eye, they decrease
intraocular pressure by suppressing the production of aqueous humor. Since the drugs are also
e ective in suppressing muscle quivering and in suppressing overreaction of the thyroid gland
upon being too nervous, they are used as doping drugs in competitive sports such as shooting
and archery, which require psychic powers of concentration rather than aerobic performance.
As untoward e ects of these drugs, chill of extremities due to the contraction of vessels and
aggravation of bronchial asthma due to contraction of the bronchi can be mentioned.
 e drugs can be classi ed into β
1
- and β
2
-blockers; the β
1
-receptors are mainly located in
the heart muscle, while the β
2
-receptors located in the smooth muscles of the airways and
blood vessels.  e β
1
-blockers speci cally exert blocking action on the β
1
-receptors; otherwise,
the β-blockers act on both β

• Mobile phases for HPLC: mobile phase A, acetonitrile/phosphoric acid/distilled water
(15:0.1:85, v/v); mobile phase B, acetonitrile/phosphoric acid/distilled water (60:0.1:40, v/v).
HPLC conditions
Separation column: Nucleosil7C
18
(250 × 4.6 mm i. d., Macherey-Nagel, Düren, Germany).
HPLC conditions; instrument: L-6200 type
b
(Hitachi Ltd., Tokyo, Japan); autosampler:
WISP 714 plus (Waters, Milford, MA, USA);  ow rate: 1.5 mL/min. Gradient elution is linearly
made from 100 % A to 100 % B during 8 min a er injection. Detector: HP1040DAD (Agilent
Technologies, Palo Alto, CA, USA); detection wavelengths: 216, 254 and 275 nm, because of
low speci city of the detection by ultraviolet absorbance.
Procedure
i. A 1-mL volume of a specimen, 50 µL IS solution (containing 5 µg ethyltheophylline) and
0.5 g of solid carbonate bu er are placed and mixed well in a glass centrifuge tube with a
ground-in stopper, followed by the addition of 5 mL diethyl ether and gentle shaking for
5 min.
ii. A er centrifugation at 1,000 g for 5 min, the ether phase is transferred to a vial and evapo-
rated to dryness under a stream of nitrogen.
iii.  e residue is dissolved in 300 µL methanol and a  xed aliquot of it is injected into
HPLC
c
.
iv. Blank specimens are spiked with various concentrations of the authentic standard of a drug
and the IS, and are processed according to the above procedure to construct each calibra-
tion curve. By applying the peak area ratio of a specimen to the calibration curve, the
concentration of a target compound is calculated.
Assessment of the method
> Figure 7.1 shows HPLC chromatograms for atenolol, nadolol and labetalol extracted from

them. The results were obtained after single oral intake of each drug. In the analysis of atenolol,
double peaks appeared for this drug, because of two asymmetrical carbon atoms present in its
structure.
⊡ Figure 7.1
Simultaneous GC/MS analysis of β-blockers and their metabolites
372 β-Blockers
Flow rate: 1 mL/min (about 20 kPa/cm
2
of He pressure at 100 °C); injection temperature:
300 °C; split ratio = 11:1; sample volume to be injected: 2 µL; ionization mode: EI; electron
energy: 70 eV; ion multiplier: auto tune + 300 V
 e range in the scan mode: m/z 50–600; dwell time of each ion in the SIM mode: 30 ms/
ion.  e measurements are made for the following 3 groups by switching them according to
retention times.
Ion group 1: from 2.5 min of retention time
m/z 86, 129, 200, 284, 365, 373, 427, 478, 479, 505, 526 and 559
Ion group 2: from 5.1 min of retention time
m/z 86, 129, 235, 250, 284, 344, 348, 421, 448, 478, 488, 497, 510 and 526
Ion group 3: from 6.0 min of retention time
m/z 86, 91, 129, 284, 292, 478 and 515.
Procedure
i. A 5-mL volume of urine, 50 µL IS solution (containing 10 µg bupranolol), 1 mL of 1 M
phosphate bu er (pH 7) and 30 µL of β-glucuronidase K-2 (3 nuits, Roche Diagnostics
GmbH, Mannheim, Germany) are placed in a glass centrifuge tube with a ground-in stop-
per, and incubated at 50 °C for 60 min for hydrolysis.
ii.  e hydrolyzed solution is mixed with 5 mL diethyl ether and gently shaken for 5 min for
washing the aqueous phase. A er removal of the organic phase, 1 mL 2-methylpropanol
and about 0.1 g of solid carbonate bu er are added to the aqueous phase and mixed well.
iii. A 5 mL volume of diethyl ether
f

representative fragment ions each, obtained from urine extracts of volunteers, who had received
single oral administration of each β-blocker under informed consent.  e metabolites hydroxy-
lated at the aromatic rings followed by its glucuronide conjugation are found in common with
most β-blockers; especially for alprenolol, metoprolol, penbutolol and propranolol, the amounts
of metabolites to be excreted into urine are larger than those of their unchanged forms.
⊡ Table 7.1
Indicators for GC/MS analysis of β-blockers
β-Blocker Compound(s)
to be detected
Derivatization Relative
retention
time*
M. W. Monitor ions
(m/z)
acebutolol acebutolol
acetylacebutolol
N-TFA-O-TMS
N-TFA-O-TMS
2.313
2.196
504
476
284
284
129
129
alprenolol hydroxyl aprenolol N-TFA-bis-O-TMS 1.618 505 284 505
atenolol atenolol N-TFA-bis-O-TMS 1.630 559 284 559
betaxolol betaxolol N-TFA-O-TMS 2.039 475 284 129
bisoprolol bisoprolol N-TFA-O-TMS 2.027 493 284 129

129
penbutolol hydroxyl penbutolol N-TFA-bis-O-TMS 1.797 451 86 250
pindolol pindolol N-TFA-bis-N,O-TMS 2.061 488 284 488
propranolol hydroxyl propranolol
4- hydroxyl propranolol
N-TFA-O-TMS
N-TFA-O-TMS
1.708
2.148
427
515
284
284
427
515
sotalol sotalol N-TFA-bis-N,O-TMS 1.851 512 344 497
timolol timolol O-TMS 1.627 388 86 373
IS bupranolol N-TFA-O-TMS 1.000 439 86
* Relative retention time: retention times of each compound, when that of IS is assumed as 1.000.
Simultaneous GC/MS analysis of β-blockers and their metabolites