Determination of Pregabalin in Human Plasma Using LC-MS-MS

Determination of Pregabalin in Human
Plasma Using LC-MS-MS
2008, 67, 237–243
Uttam Mandal, Amlan Kanti Sarkar, Kadagi Veeran Gowda, Sangita Agarwal, Anirbandeep Bose,
Uttam Bhaumik, Debotri Ghosh, Tapan Kumar Pal&
Bioequivalence Study Centre, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 32, India;
E-Mail: [email protected]
Received: 2 August 2007 / Revised: 25 September 2007 / Accepted: 9 October 2007
Online publication: 8 January 2008
Introduction
Abstract
A bioanalytical method has been developed and validated for determination of pregabalin in
human plasma. The analytical method consists in the precipitation of plasma sample with
trichloro acetic acid (20% v/v solution in water), followed by the determination of pregabalin
by an LC-MS-MS method using gabapentin as internal standard. Separation was achieved
on a Gemini C18 50 mm · 2.0 mm (3 lm) column with an isocratic mobile phase consisting
of methanol–water (98:2, v/v) with 0.5% v/v formic acid. Protonated ions formed by a turbo
ionspray in positive mode were used to detect analyte and internal standard. The MS-MS
detection was by monitoring the fragmentation of 160.2?55.1 (m/z) for pregabalin and
172.2?67.1 (m/z) for gabapentin on a triple quadrupole mass spectrometer. The assay
was calibrated over the range 0.1–15.0 lg mL 1 with correlation coefficient of 0.9998.
Validation data showed intra-batch (n = 6) CV% 6.89 and RE (%) between 4.17 and
+3.08 and inter-batch (n = 18) CV% < 9.09 and RE (%) between 3.0 and +10.00. Mean
extraction recovery were 80.45–89.12% for three QC samples and 87.56% for IS. Plasma
samples were stable for three freeze–thaw cycles, or 24 h ambient storage, or 1 and
3 months storage at 20 C. Processed sample (ready for injection) were stable up to 72 h
at autosampler (4 C). This method has been used for analyzing plasma samples from a
bioequivalence study with 18 volunteers.
Keywords
Column liquid chromatography–mass spectrometry
Bioequivalence study
Turbo ionspray
Pregabalin
Original
DOI: 10.1365/s10337-007-0440-2
0009-5893/08/02
Pregabalin (PGB), (S)-3-aminomethyl-5methyl hexanoic acid, is a structural
analogues of c-aminobutyric acid
(GABA) as shown in Fig. 1.a. It is recently approved in the European Union
(EU) as an adjunctive therapy for
treatment of partial seizures with and
without generalized tonic–clonic seizures and for the treatment of peripheral neuropathic pain in adults [1–8].
PGB binds potentially to the a2-d subunit, an auxiliary protein, of Q-type
voltage-sensitive calcium channels that
are widely distributed throughout the
peripheral nervous system and CNS
[9–11]. Potent binding at this site
reduces calcium influx at hyperexcited
nerve terminals and, therefore, reduces
the release of several neurotransmitters,
including glutamate, noradrenaline, and
substance P [12–15]. These activities and
effects result in the anticonvulsant,
analgesic, and anxiolytic activity exhibited by PGB. PGB is inactive at c-aminobutyric acid (GABA)A and GABAB
receptors; it is not converted metabolically into GABA or a GABA antagonist, and it does not alter GABA uptake
or degradation [16, 17].
Chromatographia 2008, 67, February (No. 3/4)
2008 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH
237
Table 1. Tandem mass spectrometric parameters of pregabalin and gabapentin
Compound
Mol.
Wt.
Protonated
ion
Fragment
CE
(eV)
DP
(V)
EP
(V)
FP
(V)
CXP
(V)
Dwell
time (ms)
Pregabalin
Gabapentin
159.2
171.2
160.2
172.2
55.1
67.1
35
46
19
26
10.5
10.0
398
390
1.5
2
200
200
eV Electron volt, V volt, CE collision energy, DP Declustering potential, EP entrance potential,
FP focusing potential, CXP collision cell exit potential, ms milliseconds
Experimental
Materials and Reagents
Fig. 1. Chemical structure of pregabalin and
gabapentin : a pregabalin and b gabapentin
PGB is not appreciably metabolized
in man, and studies in healthy volunteers
indicate oral bioavailability to be
approximately 90% [18]. This contrasts
with gabapentin, which is absorbed via a
capacity-limited L-amino-acid transport
system from the proximal small bowel
into the blood stream [19, 20].
There are few published methods
for analysis of pregabalin in human
plasma by HPLC after precolumn
derivatization of pregabalin using
o-phtaldialdehyde (OPA) and 3-mercaptopropionic acid [21] or picrylsulfonic acid [22]. In practical application,
derivatization may be a difficult technique and gives inaccurate estimation
of analyte due to incomplete derivatization if reaction conditions are not
strictly maintained. In comparison to
precolumn derivatization for HPLC,
our developed LC-MS-MS method is a
simple one step precipitation, it is
accurate and requires less time (2 min
only) without derivatization.
238
Pregabalin (>99%) was supplied by
Burgeon Pharmaceutical Pvt., Chennai,
India. Gabapentin (Fig. 1b) (>99%) as
internal standard (IS) was obtained from
Cosmas Pharmaceuticals, Ludhiana, India. Formic acid (98%), trichloro acetic
acid (analytical-reagent grade), methanol (HPLC grade) were purchased from
Merck Pvt., Mumbai, India. Water
(resistivity of 18 MX) was collected
from a Milli-Q gradient system of
Millipore (Elix 3, Milli-Q A10
Academic). The blank human plasma
with EDTA-K3 anticoagulant was collected from Clinical Pharmacological
Unit (CPU) of Bioequivalence Study
Centre, Jadavpur University, Kolkata,
India.
by AB Sciex Instruments (Toronto,
Canada) for detection. Sciex Analyst
software version 1.4.1 was used for data
acquisition and processing. Turbo ionspray ionization source was operated in
a positive mode for mass spectrometric
detection. The collision energy (CE) and
other parameters for the analyte and IS
were optimized by infusing each compound solution with a concentration of
500 ng mL 1 in water. The multiplereaction mode (MRM) was used to
acquire ion counts at different time
points. A high voltage of 5.5 kV was
applied to the spray needle. The instrument was programmed for a scan dwell
time of 200 ms. The source temperature
was set at 550 C, using nitrogen
(5.0 grade) at 7 L min 1 as auxiliary gas
and zero grade air as nebulizer gas at a
pressure of 80 p.s.i. (1 p.s.i. = 6894.76
Pa). The setting of curtain gas and
collision gas flow at instrument were 10,
12 (arbitrary scale), respectively. All gas
used in this experiment was generated
from a Peak gas generator (Chicago, IL,
USA). The collision energies and other
optimized parameters used for analyte
and IS are presented in Table 1. In this
method, both Q1 and Q3 quadrupoles
were operated at unit resolution. For
each injection, the total acquisition time
was 2 min.
LC-MS-MS
The LC system consisting of solvent
delivery LC 10ADVP, Controller
LC10ADVP
and
column
oven
CTO10ASVP were from Shimadzu
(Kyoto, Japan). Sample injection was
with a Shimadzu SIL HTC Autosampler. The analytical column used was a
Gemini C18 50 mm · 2.0 mm (3 lm)
from Phenomenex, USA. Elution was
achieved at room temperature with
methanol–water (98:2, v/v), 0.5% v/v
formic acid as the mobile phase. The
LC system was operated isocratically at
1 mL min 1. The column eluent was
split and approximately 400 lL were
introduced in the mass spectrometer.
The total run time for each sample
analysis was 2 min only.
The LC system was coupled with a
turbo ionspray ionization-triple quadrupole mass spectrometer API 2000 made
Standard Solutions and
Quality Control (QC) Samples
The stock solutions of analyte and IS
were prepared at 1 mg mL 1 in water
by vortexing approximately 1 min.
Dilutions of 100 and 10 lg mL 1 were
made from the stock solutions which
were used to prepare the calibration
curve and quality control (QC) samples.
An eight-point standard curve was
prepared by spiking the blank plasma
with appropriate amounts of working
solution to obtain final concentrations
of 0.1, 0.25, 0.5, 1, 2.5, 5, 10 and
15 lg mL 1 for the analyte. The
concentration of IS in plasma sample
was 5.0 lg mL 1. All stock solutions
and working standard solutions were
stored in polypropylene vials in a
20 C freezer.
Chromatographia 2008, 67, February (No. 3/4)
Original
4.3e5
160.2
(a)
182.1
4.0e5
3.5e5
3.0e5
Intensity (cps)
The linear regression of the peak area
ratio of analyte/IS versus concentration
using a weighed 1/concentration2 was
used to obtain the calibration curve. The
regression equation of the calibration
curve was then used to calculate the
plasma concentration. The back calculated values of the concentrations were
statistically evaluated.
Quality control samples were made
using the stock solution. Four levels of
QC samples in plasma were 0.050 (lower
limit of quantitation, i.e. LLOQ), 0.3
(low-), 6.0 (medium-), and 12.0 (high-)
lg mL 1 for the analyte. QC samples
were prepared in a 50 mL pool then
aliquoted into pre-labeled 2 mL polypropylene vials and stored at 20 C.
2.5e5
2.0e5
119.3
201.2
1.5e5
142.2
1.0e5
161.0
199.2
151.0
171.1 176.9
183.0
204.0
143.2
157.9 164.9
141.1
104.9
200.2
180.9
129.0
147.1
179.0
184.9
108.9 115.9 125.2
205.1
193.0
173.0
145.1
164.0
149.9155.0
132.0136.2
188.8
107.1
114.0
5.0e4
149.0
117.2
130.9
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210
m/z (amu)
172.0
2.6e5
Sample Preparation
Method Validation
The method was validated for selectivity,
linearity, precision, accuracy, recovery
and stability according to the principles
of the Food and Drug Administration
(FDA) industry guidance [23]. Three
validation batches were processed on
three separate days. Each batch included
one set of calibration standards and six
replicates of LLOQ, low-, medium-, and
high-concentrations of QC samples.
Inter-batch and intra-batch precision
Original
2.4e5
2.2e5
2.0e5
1.8e5
Intensity (cps)
For calibration standards, an aliquot of
0.1 mL for each spiking solution was
spiked into 0.9 mL of control blank
plasma in polypropylene tube. Then
0.1 mL of IS working solution was added to each tube and all the samples
were vortex-mixed for 30 s. Then 0.2 mL
of trichloro acetic acid (20% v/v solution
in water) was added followed by 10 min
mixing by cyclo mixer. All the samples
were centrifuged for 15 min at
5,000 rpm. The supernatant clear solution was separated and filtered through a
0.45 lm membrane filter. The resulting
samples were transferred into a 1.0 mL
glass vial which was loaded into the
autosampler cabinet and 20 lL aliquot
of each extracted sample was injected
into the LC-MS-MS system.
(b)
1.6e5
149.0
1.4e5
1.2e5
153.9
1.0e5
119.0
8.0e4
6.0e4
4.0e4
2.0e4
176.9
180.9
130.9
160.9
137.2
176.0
129.1
115.2
162.9
173.0
151.0 156.9
117.1
178.9
105.1
147.0
113.1
127.9
114.1
180.0 183.1
145.0
159.9 166.9
121.1
134.9139.1
185.0
149.9 157.9
106.2
170.9
145.9
131.9
112.2
120.2123.8125.0
167.9
103.1
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190
m/z (amu)
Fig. 2. Parent ion mass spectra: a pregabalin (m/z 160.2) and b gabapentin (m/z 172.2)
and accuracy evaluations were based on
back-calculated concentrations.
The selectivity is the ability of an
analytical method to differentiate and
quantify the analyte in the presence of
other components in the sample. This
test was performed by analyzing the
blank plasma samples from different
sources (or doners) to test for interference at the retention time of pregabalin
and gabapentin (IS).
The recovery of pregabalin (low-,
medium- and high-QC) and gabapentin
(5.0 lg mL 1) from human plasma were
evaluated by comparing the peak area
response of precipitated analyte and
internal standard with that of reference
solutions at the same concentration level
and reconstituted into blank plasma extracts. The number of replicates for each
concentration was six.
Stability study was evaluated as part
of the method validation. The processed
sample stability was evaluated by comparing the precipitated samples that were
injected immediately (time 0), with the
samples that were re-injected after loading into the autosampler at 4 C for 72 h.
QC samples were kept at ambient temperature for 24 h and analyzed against
freshly spiked standard curve and QC
samples for short-time stability. The longterm stability of spiked human plasma
stored at
20 C was evaluated by
Chromatographia 2008, 67, February (No. 3/4)
239
pregabalin sample time and to infinity
(AUC0-t and AUC0-inf), maximum concentration (Cmax), time to maximum
concentration (tmax), elimination rate
constant (Kel) and elimination half-life
(t1/2) were determined for the period of
0–24 h by non compartmental method.
Results and Discussion
Mass Spectrometry
LC-MS-MS for the determination of
pregabalin in human plasma was investigated. Positive electrospray mass spectra of pregabalin shows an intense
[M + H] + ion at m/z 160.2 (Fig. 2a).
Another intense [M + H]+ ion at m/z
172.2 is shown for gabapentin (Fig. 2b).
When these molecular ions undergo
fragmentation in the collision cell, the
product ion mass spectra shown in Fig. 3a
and b are generated. With the experimental conditions used in these experiments,
pregabalin shows an intense product ion at
m/z 55.1 which corresponds to the positively ionized fragment given below:
Fig. 3. Product ion mass spectra: a pregabalin (m/z 55.1) and b gabapentin (m/z 67.1)
analyzing all QC samples that were stored
at 20 C for 1 and 3 months together
with freshly spiked standard curve and
QC samples. The freeze–thaw stability
was conducted by comparing the stability
samples that had been frozen and thawed
three times, with the plasma samples
thawed once. Three aliquots of each QC
sample were used for freeze–thaw stability
evaluation. All stability evaluations were
based on back-calculated concentrations.
Application
The above-mentioned validated method
was successfully used to analyze plasma
samples for a bioequivalence study of
240
pregabalin. The study was approved by
the ethics committee of Jadavpur
University, Kolkata, India. It was an
open, randomized crossover study to
determine relative bioavailability of
pregabalin in eighteen healthy male
volunteers following single dose administration of pregabalin 300 mg capsule.
Test preparation was pregabalin 300 mg
capsule manufactured by Burgeon
Pharmaceutical Pvt., Chennai, India.
Capsule Newgaba containing 300 mg of
pregabalin, manufactured by Sun
Pharma Industries Pvt., Mumbai, India
was used as reference preparation. The
pharmacokinetic parameters like area
under the plasma-concentration–time
curve from zero to the last measurable
The product ion mass spectrum of
gabapentin shows an intense fragmentation at m/z 67.1 that corresponds to an
ionic fragment shown below:
Separation and Specificity
Typical MRM chromatograms from the
study of pregabalin and gabapentin in
human plasma are shown in Fig. 4b.
Retention time of pregabalin and IS are
at 0.79 and 0.53 min, respectively. No
interference peak was found in the
MRM profiles for six blank plasma
samples (Fig. 4a).
Chromatographia 2008, 67, February (No. 3/4)
Original
Limit of Quantitation,
Linearity
(a)
Intensity (cps)
Lower limit of quantitation was established as 50 ng mL 1, its precision
(CV%) and accuracy (%RE) values
being 10.59 and 9.83%, respectively.
The equation of the calibration curve
was obtained by least-squares linear
regression analysis of the peak-area
ratios of pregabalin to internal standard
versus concentration. The curve was
linear in the concentration range 0.10–
15.00 lg mL 1 with regression coefficient of 0.9998 (calibration equation,
y = 0.192x + 0.0158). The average
correlation coefficient was 0.9998. All
back calculated values showed excellent
accuracy and precision (Table 2). No
single calibration standard point was
dropped during the validation.
Time (min)
2.1e5
0.79
(b)
PREGABALIN
2.0e5
1.8e5
1.6e5
Precision and Accuracy
Intensity (cps)
1.4e5
The precision and accuracy of QC samples are presented in Table 3. For low-,
medium-, and high-QC samples validation data showed the intra-batch (n = 6)
CV% 6.89 and RE (%) between
4.17 and +3.08 and inter-batch
(n = 18) CV% < 9.09 and RE (%)
between 3.0 and +10.00. These results
indicate that the method was reliable
within the analytical range.
GABAPENTIN(I.S.)
1.2e5
0.53
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Time (min)
Recovery
Six replicates of three QC samples were
prepared for recovery determination.
Mean (CV%) absolute recovery for pregabalin at 0.30, 6.00 and 12.00 lg mL 1
were 80.45% (6.11), 83.19% (4.26) and
89.12% (3.08), respectively. Mean
(CV%) recovery of internal standard
(5.00 lg mL 1) was 87.56% (4.92).
Stability
Each stability test included six replicates
of three levels of QC samples. All stability results, as well as the linear
regression correlation coefficients of
calibration curves generated from each
Original
Fig. 4. Representative MRM chromatogram of pregabalin and gabapentin (IS): a blank human
plasma; b Plasma sample of a volunteer showing separation of pregabalin (0.79 min) and
gabapentin (IS) (0.53 min) after oral administration of capsule containing 300 mg of pregabalin
Table 2. Summary of calibration standards
Conc. added (lg mL 1)
0.10
0.25
0.50
1.00
2.50
5.00
10.00
15.00
Conc found (lg mL 1)
SD
CV (%)
RE (%)
n
0.11
0.24
0.53
0.93
2.63
5.15
9.75
14.41
0.01
0.02
0.04
0.06
0.15
0.39
0.4 9
0.68
9.09
8.33
7.55
6.45
5.70
4.82
5.02
4.72
+10.00
4.00
+6.00
7.00
+5.20
+3.00
2.50
3.93
6
6
6
6
6
6
6
6
SD Standard deviation, CV (%) coefficient of variation [(SD/Mean) · 100], RE (%) relative
error [{(Conc. Found Conc. Added)/Conc. Added} · 100], n number of replicates
stability test run for the analyte are
presented in Table 4. QC samples
undergoing three freeze–thaw cycles
gave % CV 6.41 and an accuracy of
92.81–98.27%. QC samples storing at
ambient for 24 h gave 5.75% CV and
Chromatographia 2008, 67, February (No. 3/4)
241
Application
Table 3. Precision and accuracy for pregabalin
Conc. added (lg mL 1)
Intra-batch
0.30
6.00
12.00
Inter-batch
0.30
6.00
12.00
Conc found (lg mL 1)
SD
CV (%)
0.29
5.75
12.37
0.02
0.31
0.53
6.89
5.39
4.28
3.33
4.17
+3.08
6
6
6
0.330
5.92
11.64
0.03
0.35
0.72
9.09
5.91
6.19
+10.00
1.33
3.00%
18
18
18
RE (%)
n
SD Standard deviation, CV (%) coefficient of variation [(SD/Mean) · 100], RE (%) relative
error [{(Conc. Found Conc. Added)/Conc. Added} · 100], n number of replicates
Table 4. Short-term and long-term stability data
Storage Condition
Low-QC
(0.300 lg mL 1)
Medium-QC
(6.0 lg mL 1)
High-QC
(12.0 lg mL 1)
r2
3 Freeze/thaw cycle
24 h ambient
72 h at autosampler
1 month frozen ( 20 C)
3 month frozen ( 20 C)
92.81
93.61
96.49
94.23
92.29
96.43
96.18
97.52
97.11
96.41
98.27 (5.25)
97.84 (2.23)
100.19 (4.72)
98.45 (1.97)
97.01 (2.40)
0.9998
0.9982
0.9978
0.9995
0.9989
(6.41)
(5.75)
(2.68)
(6.19)
(6.87)
(3.17)
(3.87)
(2.98)
(4.42)
(3.66)
The data presented in this table are the percentage of measured value vs. theoretical value with
CV in parenthesis (n = 6). r2 is the linearity of the calibration curve used for this treatment
The
above-mentioned
LC-MS-MS
method was used in the plasma sample
analysis for a bioequivalence study of
pregabalin as described above. Figure 5
shows the mean (±SD) plasma level of
pregabalin for test and reference preparation after the oral administration of a
single dose 300 mg capsule of pregabalin
in 18 healthy human volunteers. Maximum plasma concentration (Cmax) ranged from 6.925 to 7.477 lg mL 1 at
1.86–2.12 h (tmax). The elimination half
life (t1/2) ranged from 3.99 to 5.33 h with
elimination rate constant (Kel) of 0.096–
0.185. Also the mean value of area under
the concentration time curve (AUC0-t)
obtained was 43.079–48.768 lg h mL 1
and AUC0-inf was found to be 45.252–
57.446 lg h mL 1. Relative bioavailability of test preparation was 93.67% to
that of reference preparation and both
the products were bioequivalent.
Conclusions
Fig. 5. Mean (±SD) plasma concentration of pregabalin following 300 mg oral dose of test and
reference preparation to 18 healthy human volunteers
an accuracy of 93.61–97.84%. Processed
samples (ready for injection) were found
to be stable for at least 72 h at 4 C in
the autosampler with %CV of 4.72%
and accuracy of 96.49–100.19%.
Long term frozen storage stability
was tested at 1 and 3 month after QC
sample pools were prepared and stored
242
at 20 C. The 1-month stability data of
all three QC samples showed an accuracy of 94.23–98.45% (CV% 6.19) in
comparison with their theoretical values
in plasma samples. The 3-month stability
data of all three QC samples had an
accuracy of 92.29–97.01% (% CV
6.87) in plasma.
The LC-MS-MS method described here
has significant advantages over the other
techniques already described in the
literature [21, 22]. The method has
proved to be fast with each sample
requiring a run time of 2 min only and
therefore has a high throughput capability. The assay method is specific due
to the inherent selectivity of tandem
mass spectrometry. The major advantage of this method is the simple one step
precipitation for sample preparation.
The proposed method to analyze
pregabalin in plasma by LC-MS-MS
method happens to be first of its kind
described so far in the literature. This
new method will be helpful for carrying
out pharmacokinetic study.
Acknowledgments
The authors are thankful to Burgeon
Pharmaceutical Pvt. Ltd., Chennai,
India, and Cosmas Pharmaceuticals,
Ludhiana, India for supplying the gift
samples of pregabalin and gabapentin
Chromatographia 2008, 67, February (No. 3/4)
Original
respectively. The authors also acknowledge the Department of Science and
Technology (DST), Govt. of India, New
Delhi, for providing financial assistance
to carry out this project through their
project No. VII-PRDSF/56/05-06 under
DPRP programme.
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