ACTA CHROMATOGRAPHICA, NO.

19, 2007

A VALIDATED HPLC METHOD FOR ESTIMATION

OF METOPROLOL IN HUMAN PLASMA

M. Aqil1,*, A. Ali1, A. Ahad1, Y. Sultana1, A. K. Najmi1, and N. Saha2

1
Faculty of Pharmacy, Hamdard University New Delhi, 110 062, India
2
Ranbaxy Clinical Pharmacology Unit, Majeedia Hospital,
New Delhi 110 062, India

SUMMARY
A high-performance reversed-phase liquid chromatographic method
for quantification of metoprolol tartrate (MT) in human plasma is presen-
ted. A C18 column was used with acetonitrile–water–triethylamine 18:81:1
(v/v) as mobile phase and pinacidil monohydrate as internal standard (IS).
UV detection was at 275 nm and MT and the IS were detected at retention
times of 1.5 and 2.6 min, respectively. The method is sensitive with a li-
mit of quantification of 20 ng mL−1. The calibration plot for MT in spiked
plasma was linear in the concentration range 20–200 ng mL−1. Within-batch
and total accuracy of the method ranged between 99.71% and 101.61%,
and within-batch and total precision, expressed as the coefficient of varia-
tion, was 0.20–2.13%. Recovery of MT from spiked plasma was 97.00%
and the freeze–thaw and bench-top stability of samples ranged from 77.92
to 105.62%. The method can be successfully used for analysis of MT in
human plasma during pharmacokinetic studies.

INTRODUCTION
Metoprolol tartrate (MT) is an established selective β1-blocking drug
used for management of moderate to severe essential hypertension [1]. Ma-
ny dosage forms of MT are available commercially, including tablets. Be-
cause the drug undergoes hepatic first-pass metabolism in the body, leading
to low oral bioavailability, a transdermal drug-delivery system has been de-
veloped to circumvent the problem. This work is part of clinical studies per-
formed to ascertain the comparative bioavailability of MT from oral and
transdermal dosage forms.
Several methods have been reported for quantification of MT in
plasma using high-performance liquid chromatography (HPLC) with UV

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or fluorescence detection [2–14]. Many of these methods involve a com-
plex separation step and are non-reproducible. This paper reports a simple,
rapid, sensitive, and reproducible method for analysis of MT in plasma,
using pinacidil monohydrate as internal standard (IS). The method was va-
lidated for accuracy, precision, linearity, sensitivity, and stability.

EXPERIMENTAL
Chemicals and Reagents
Metoprolol tartrate MT (Fig. 1) was a gift from Astra-IDL, India.
Pinacidil monohydrate PM was obtained from Leo Pharmaceuticals, Den-
mark. HPLC-grade acetonitrile and water were from Merck, India. All other
chemicals and solvents were HPLC or AR-grade and were used as received.

OH
OCH 2CHCH 2NHCH(CH 3)2 COOH
CHOH
CHOH
COOH
CH 2CH 2OCH 3
2
Fig. 1
The chemical structure of metoprolol tartrate

MT working standard (5 mg) was accurately weighed, transferred

to a 50-mL volumetric flask, and diluted to volume with the mobile phase
to produce a stock solution containing 100 µg mL−1.
A solution of PM, the internal standard (IS), was prepared by dis-
solving 5 mg PM in mobile phase in a 50-mL volumetric flask and diluting
to volume with the mobile phase to furnish a stock solution containing 100
µg mL−1. For spiking plasma samples this stock solution was diluted to 2
µg mL−1 with water.
Chromatography
HPLC was performed with a Shimadzu (Japan) SCL10AT system
comprising an LC-10 AT pump, an autosampler, and an SPD-10A detec-

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tor. Data processing was by Shimadzu Class-VP software on a Hewlett–Pa-
ckard computer. Compounds were separated on a 250 mm × 4 mm, 10-µm
particle, Novapack C-18 column.
Before method development, column performance was tested by
duplicate injection of a mixture of resorcinol, naphthalene, and anthracene
(10 µL) with acetonitrile–water 55:45 as mobile phase at a flow rate of 1.0
mL min−1. Detection was by UV absorption at 254 nm. System suitability
was then tested by injecting a dilute solution of drug five times using the
same chromatographic conditions.
The mobile phase used for the method was acetonitrile–water–tri-
ethylamine 18:81:1 (v/v), pH adjusted to 11 by use of phosphoric acid; be-
fore use the mobile phase was degassed and filtered through a 0.45-µm fil-
ter. The flow rate was 1.0 mL min−1 and MT was detected by UV absorp-
tion at 275 nm.
Calibration
Serial dilutions (1–10 µg mL−1) of the MT stock solution were pre-
pared in the mobile phase. Each of these dilutions (1 mL) was added to 49
mL screened plasma, to furnish concentrations in the range 20–200 ng mL−1.
These samples were analysed by HPLC and the areas of the MT peak we-
re plotted against the concentration of MT. The calibration data obtained
are listed in Table I and the calibration plot constructed from the data is
shown in Fig. 2.

Table I
Calibration data for metoprolol tartrate in water–acetonitrile–triethylamine 81:18:1 (v/v)
after spiking of plasma with stock solutions

Drug concentration Peak Regressed value

No. SD
in spiked plasma (ng mL−1), X areaa of peak area, Y
1 20 154694 165856 3218
2 40 298620 303284 2873
3 80 596181 578141 1528
4 120 865487 852997 4384
5 160 1117645 1127853 3024
6 200 1398217 1402710 5387
a
n = 5, i.e. results are means from five observations

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 1600000
1400000
1200000
Peak area

1000000
800000
600000
400000
200000
0
0 20 40 60 80 100 120 140 160 180 200
Drug conc. in spiked plasma (ng/ml)

Fig. 2
Calibration plot for metoprolol tartrate in water–acetonitrile–triethylamine 81:18:1 (v/v)
after spiking of plasma with the pure drug. Regression equation, Y = 6871X +28428, cor-
relation coefficient, r, 0.9997

Method Validation
The method was validated for accuracy, precision, sensitivity, reco-
very, linearity, and stability in accordance with ICH guidelines [15], using
PM as IS. Linearity, precision, and accuracy were tested by analysis of
five batches of spiked plasma quality-control (QC) samples. Each batch of
spiked plasma samples included one complete set of calibration standards
(comprising six different concentrations, blank, and blank with internal stan-
dard) and QC samples at low (40 ng mL−1, LQC), middle (70 ng mL−1,
MQC), and high (100 ng mL−1, HQC) concentrations.
Linearity was determined by regression analysis of the calibration
data and determination of the coefficient of variation. Sensitivity was de-
termined as the limits of detection (LOD) and quantification (LOQ). The
accuracy of the method was evaluated as the MT-to-IS peak-area ratio.
Precision was measured as the coefficient of variation (%) for the low, mid-
dle, and high QC samples during validation. Recovery (%) was determined
by measuring the MT-to-IS peak-area ratio for the plasma QC samples
(LQC, MQC, and HQC) with the MT-to-IS peak-area ratios of freshly pre-
pared unextracted aqueous standards containing the same concentrations of
MT. To determine the freeze–thaw stability of spiked plasma samples five
replicates each of the low, middle, and high QC samples were analyzed
after one and two freeze–thaw cycles. The freeze–thaw QC samples were

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quantified by comparison with a calibration plot prepared after chromato-
graphy of freshly spiked samples. For assessment of bench-top stability,
the concentrations of low, middle, and high QC samples were determined
after 0, 2, and 4 h. The MT-to-IS peak-area response ratios after 2 and 4 h
were compared with that after 0 h.

RESULTS AND DISCUSSION

Under these chromatographic conditions MT and IS in a plasma
matrix were resolved with retention times (tR) of 1.5 and 2.6 min, respec-
tively. The method was found to be suitable because reproducible results
were obtained on each sample run. On validation of the above method using
PM as the IS, the following results were obtained.
Linearity
The linearity of the calibration plot for MT in human plasma at
concentrations 20, 40, 80, 120, 160, and 200 ng mL−1 was excellent
1.567
Volts

2.683

Minutes

Fig. 3
Representative chromatogram obtained during quantification of metoprolol tartrate (tR = 1.5
min) in human plasma with pinacidil monohydrate (tR = 2.6 min) as IS

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(r = 0.9997) over the concentration range investigated. A typical calibration
plot obtained during plasma analysis could be described by the linear
equation, Y = 6871X + 28428, where Y is peak area and X is concentration
(ng mL−1). Five calibration plots were constructed; the results obtained are
presented in Table I and Figs 2 and 3.
Sensitivity
The limits of detection (LOD) and quantification (LOQ) for me-
toprolol tartrate were calculated by use of the formulae LOD = 3.3σ/S and
LOQ = 10σ/S, where σ is the standard deviation of the response and S is
the slope of the calibration plot. For analysis of 1 mL spiked plasma the
LOD was 5 ng mL−1 and the LOQ 20 ng mL−1.
Accuracy
The within-batch accuracy, determined by use of the internal stan-
dard area-ratio method, ranged from 99.71 to 101.61% (Table II). The be-
tween-batch or total accuracy, determined by use of the internal standard
area-ratio method, was found to be 99.91 to 100.60% (Table II).

Table II
Within and between-batch precision and accuracy for analysis of metoprolol tartrate

Nominal concentration (ng mL−1)

LQCa, MQCa, HQCa,
Actual
40.4721 70.5232 100.7328
QC1 40.3625 70.8324 100.3467
QC2 40.6823 70.5983 100.5675
QC3 39.9847 71.7345 100.8635
QC4 41.2839 69.2384 99.9928
Mean 40.5783 70.6009 100.4426
SD 0.5500 1.032 0.3671
CV (%) 1.35 1.46 0.36
Nominal (%) 100.26 100.01 99.71
QC5 40.4683 70.9395 100.9338
QC6 39.8341 69.7682 99.9853
QC7 40.7375 70.7555 100.752
QC8 41.8382 70.3248 100.3253
Mean 40.7195 70.4470 100.4992
SD 0.8364 0.5207 0.4271
CV (%) 2.05 0.74 0.42
Nominal (%) 100.61 99.89 99.77

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Table II (continued)
Within and between-batch precision and accuracy for analysis of metoprolol tartrate

Nominal concentration (ng mL−1)

QC9 40.5354 70.6848 100.9461
QC10 40.8252 69.5347 100.7877
QC11 39.8411 70.3548 100.8089
QC12 40.7231 71.4039 100.4040
Mean 40.4812 70.4945 100.7366
SD 0.3839 0.6715 0.2015
CV (%) 0.95 0.95 0.20
Nominal (%) 100.02 99.96 100.00
QC13 40.1001 70.3845 101.3801
QC14 39.9387 69.9128 100.9821
QC15 41.8177 70.6599 100.5347
QC16 40.9462 70.6848 100.7081
Mean 40.7007 70.4105 100.9013
SD 0.8659 0.3586 0.3685
CV (%) 2.13 0.51 0.36
Nominal (%) 100.56 99.84 100.17
QC17 40.4076 71.9123 100.5157
QC18 41.5347 70.8652 101.3738
QC19 40.9995 69.3920 100.6598
QC20 41.4696 70.5830 99.9991
Mean 41.1028 70.6881 100.6371
SD 0.5212 1.0362 0.5672
CV (%) 1.27 1.46 0.56
Nominal (%) 101.56 100.23 99.90
Total global statistics
LQC, MQC, HQC,
Actual
40.4721 70.5232 100.7328
Mean 40.7165 70.5282 100.6434
SD 0.6147 0.7119 0.3994
CV (%) 1.51 1.01 0.40
Nominal (%) 100.60 100.00 99.91
a
LQC, low quality control; MQC, middle quality control; HQC, high quality control

Precision
Within-batch precision, determined by use of the internal standard
area-ratio method, ranged from 0.20 to 2.13% (Table II). The between-batch

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or total precision, determined by use of internal standard area ratio method,
ranged from 0.40 to 1.15% (Table II).
Recovery
Recovery (%) was determined by comparing the MT-to-IS peak-area
ratio for the plasma QC samples (LQC, MQC, and HQC) with the MT-to-IS
peak-area ratio for freshly prepared unextracted aqueous standards contai-
ning the same concentrations of MT. Total recovery of metoprolol tartrate
was 93.95% and within batch recovery was 91.59 to 97.00% (Table III).

Table III
Absolute recovery of metoprolol tartrate from human plasma

Individual % recovery Total

QC
LQC MQC HQC recovery (%)
1 91.5345 95.7266 97.4632
2 99.6366 88.7573 94.3177
3 90.3233 95.4163 94.7687
4 87.4822 90.5341 98.9154
5 88.9958 95.9528 99.5640
Mean 91.5945 95.2774 97.0058 93.9592
SD 4.7423 2.9701 2.3786 4.0968
CV (%) 5.18 3.18 2.45 4.36
n 5 5 5 15

Stability
The freeze–thaw stability ranged from 77.92 to 103.02%. The sta-
bility through one and two freeze–thaw cycles was from 85.06 to 103.02%
and from 77.92% to 89.76%, respectively. The freeze–thaw stability of MT
over two cycles was therefore acceptable (Table IV). To meet acceptance
criteria MT must be stable in plasma samples for up to 4 h. The bench-top
stability ranged between 88.74 to 105.62% (Table V).
The results from method validation are summarised in Table VI. The
method was used for quantification of MT in plasma to investigate compa-
rative bioavailability after administration of the drug by the oral and trans-
dermal routes [16]. Experimental values of pharmacokinetic data after oral
administration were comparable with literature data [1].

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Table IV
Freeze–thaw stability of metoprolol tartrate in human plasma

Individual stability (%)

LQC MQC HQC
One cycle
1 91.3628 99.8230 99.7645
2 78.7324 95.1231 103.2015
3 83.7189 97.3842 110.3246
4 86.4112 89.5673 98.7843
Mean 85.0563 95.4744 103.0187
SD 4.56 3.79 4.5257
CV (%) 5.37 3.97 4.39
n 4 4 4
Two cycles
1 85.1245 86.7321 91.8005
2 73.1911 88.2345 93.1295
3 72.8612 82.2265 88.8056
4 80.5162 79.3082 85.3098
Mean 77.9232 84.1253 89.7613
SD 5.1623 3.5528 3.0097
CV (%) 6.62 4.22 3.35
n 4 4 4

Table V
Bench-top stability of metoprolol in human plasma

% Initial
Time (h)
LQC MQC HQC
0 100.00 100.00 100.00
2 99.71 105.62 88.74
4 101.57 92.75 103.64
n 3 3 3

CONCLUSION
A simple, rapid, reproducible, and sensitive HPLC method has been
developed for analysis of MT in human plasma. The short chromatographic
run time of only 1.5 min makes this method suitable for processing of ma-

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Table VI
Summary of results from method validation

Method characteristic Result

Analyte Metoprolol tartrate
Biological Matrix Human plasma
Detection UV, 275 nm
Retention time (min)
MT 1.5
IS 2.6
Linear range (ng mL−1) 20–200
Limit of quantification (ng mL−1) 20
Limit of detection (ng mL−1) 5
Accuracy (%)
Within batch 99.71 to 101.61
Total 99.91 to 100.60
Precision (%)
Within batch 0.20 to 2.13
Total 0.40 to 1.51
Recovery (%)
Within batch 91.59 to 97.00
Total 93.95
Stability (%)
Freeze–thaw (1 cycle ) 85.06 to 103.02
Freeze–thaw (2 cycles) 77.92 to 89.76
Bench-top 88.74 to 105.62
Validation was performed in accordance with ICH guidelines [15]

ny samples in limited time for pharmacokinetic and bioequivalence studies.

The method was validated for analysis of MT in human plasma over the
range 20 to 200 ng mL−1.

ACKNOWLEDGEMENTS
The authors are grateful to Ranbaxy Clinical Pharmacology Unit,
Majeedia Hospital, Hamdard University, New Delhi for providing facilities
for this work.

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