chapter: 4 analysis of tricyclic and nontricyclic...
TRANSCRIPT
CHAPTER: 4
Analysis of Tricyclic and Nontricyclic
Antidepressants by MEPS-LC-UV/GC-MS
169
4.1. Introduction
Clomipramine, imipramine, amitriptyline, mirtazapine and citalopram are
psychoactive drugs and classified as antidepressants (Fig. 4.1). These drugs are used to
cure major depression and also used as anxiolytic agents.
N
N
CH3
CH3
Cl N
N
CH3
CH3 N
CH3
CH3
O
N
CH3CH3
N
Clomipramine Imipramine Amitriptyline
Mirtazapine Citalopram
N
N
N
CH3
Fig. 4.1 Chemical structures of the studied antidepressants
At present, there is a constant need for the development of faster and more selective
sample clean-up procedures, using small amounts of biological samples. Current
developments of sample-handling techniques are directed towards automatization and on-
170
line coupling of sample preparation units and detection systems. In addition, there is a
trend towards development of more selective sorbents for sample clean up and enrichment.
The microextraction by packed sorbent (MEPS) is a novel technique for miniaturized
solid-phase extraction (SPE) that can be connected directly to liquid or gas
chromatography without any modification [1, 2]. Compared to SPE or liquid-liquid
extraction (LLE), MEPS significantly reduce sample preparation time and organic solvent
consumption. MEPS is fully automated and a much higher recovery can be obtained with
this. It can work with sample volumes as small as 10 μL. The commercially available
presentation of MEPS uses the same sorbents as conventional SPE columns and so it is
suitable for use with most of the existing methods. The key aspect of MEPS is that the
solvent volume used for the elution of the analytes is of a suitable order of magnitude to
interface with LC-UV and GC-MS to provide a completely automated MEPS-LC or
MEPS-GC-MS system. This new technique is very promising because it is fast, simple and
it requires very small volume of samples to produce comparable results to conventional
SPE technique. MEPS was applied for the determination of local anesthetics and their
metabolites [3-12], anticancer drugs cyclophosphamide, busulfan and AZD3409 [13-15],
anti-depressant drugs [16, 17], β-blocker drugs acebutolol and metoprolol [18],
oxcarbazepine and its metabolites [19], methadone [20], methamphetamine &
amphetamine [21], metabolites of monoterpenes [22] and cocaine & its metabolites [23]
from biological samples such as plasma, urine or blood.
A number of LC-UV methods for determining the antidepressants and their
metabolites have been developed and reviewed [24-33]. For the sample preparation prior to
the LC-UV analysis of antidepressants in biological fluids, either liquid-liquid extraction
[34, 35] or solid-phase extraction [36, 37] was used. Several GC-MS methods have also
171
been reported for the determination of tricyclic and nontricyclic antidepressants in
biological fluids and environmental samples for their analytical determination [38-41].
To our knowledge, no MEPS-LC method has been described to determine
amitriptyline, imipramine, clomipramine, mirtazapine and citalopram in one run. The aim
of present study was to develop and validate an innovative analytical method for the
analysis of this group of antidepressant drugs in urine and plasma samples utilizing MEPS
as sample preparation technique with LC-UV and GC for the first time.
4.2. Experimental
4.2.1. Standards and Reagents
Standard samples of amitriptyline, imipramine, clomipramine, mirtazapine and
citalopram were purchased from Sigma Aldrich (Steinheim, Germany). LC grade methanol
was obtained from Rankem (New Delhi, India). Water was distilled after deionisation
(Riviera, SCHOTT DURAN, Mainz, Germany) and filtered using 0.45 μm Nylon 6, 6
membranes (Rankem, New Delhi India). Stock solutions (1 mg mL-1
) of amitriptyline,
imipramine, clomipramine, mirtazapine and citalopram were prepared in methanol. From
the stock solution of these drugs, a stepwise dilution series were made in water.
4.2.2 Chromatographic Systems
4.2.2.1. LC-UV Instrumentation and Conditions
The LC-UV system consisted of a Dionex P680 pump (Mumbai, India) with four
solvent chambers, a Dionex Acclaim 120 C18 column (4.6 × 250 mm; 5 m), Dionex
UVD170U detector operated at a wavelength 240 nm connected to a computer loaded with
Chromeleon software for data acquisition. Separations were carried out at room
temperature maintained at 20-22 °C. Aqueous and non-aqueous solvents were filtered with
0.45 µm Nylon 6, 6 membrane filters. Double beam UV-visible spectrophotometer SL-164
172
ELICO (Hyderabad, India) was used for obtaining the absorption spectra of these drugs.
The final optimized conditions were isocratic flow of acetate buffer (0.02 M, pH 4.2):
methanol in the ratio of 30:70 (v/v) at a flow rate of 0.9 mL min-1
.
4.2.2.2. GC-MS Instrumentation and Conditions
Gas chromatographic - mass spectrometric (GC-MS) system with model GC-MS-
QP2010 Plus (Shimadzu Corporation, Kyoto, Japan) was used for the analysis. The
capillary column used in the GC was Rtx-1 MS (30 m × 0.25 mm ID; 0.25 μm, particle
size) supplied by Restek U.S. (Bellefonte, PA, U.S.A.). Chromatographic data were
collected and recorded by GC-MS real time analysis software. Sample injection was done
in split mode (split ratio 10:1). Helium was used as the carrier gas at a flow rate of 1 mL
min-1
. The GC injector temperature was set at 270 °C. The column oven temperature was
optimized to hold at 100 °C for 1 min and then to increase by 10 °C min-1
up to 200 °C, then
increased by 15 °C min-1
up to 260 °C and then by 30 °C min-1
up to 300 °C. Mass
spectrometry conditions were as follows: electron ionization source set at 70 eV, MS
source temperature 200 °C and solvent cut time was 3.5 min. The mass spectrometer was
run in full scan mode (m/z 20-500) and in selected ion monitoring (SIM) mode at m/z 58,
85 and 195. The quantitation of samples was done by using the SIM mode. Total runtime
was 30.33 min.
4.2.3. Preparation of Biological Samples
Blood (3 mL) and urine (10 mL) specimens were obtained from the healthy
volunteers (6 persons). Blood samples were stored in separate glass tubes containing
ethylenediaminetetraacetic acid (EDTA) as the anticoagulant, and then centrifuged (within
2 h from collection) at 4000 rpm for 10 min at 5 °C. The supernatant (plasma) was then
transferred to polypropylene tubes and stored at -4 °C. These plasma samples were stable
173
over a period of 6 months. Before use, the plasma was thawed at room temperature and
centrifuged at 4000 rpm for 5 min. Spiked plasma samples were prepared by adding a few
micro liter of the analytes to 1 ml of centrifuged plasma. After that, the samples were
extracted and analyzed. The concentration range of the standard curve was between 1 and
500 ng mL-1
.
Urine samples were collected from the healthy people and stored frozen at -4 °C in
glass tubes until the time of sample pre-treatment, when they were centrifuged at 4000 rpm
for 10 min at 5 °C, the assays were carried out on the clear supernatant. Spiked urine
samples were prepared using the same procedure as described above for plasma samples.
4.2.4. MEPS Conditions
MEPS was carried out on a BIN (Barrel Insert and Needle Assembly) containing 4
mg of solid-phase silica-C18 material, inserted into a 250 μL gas-tight syringe from SGE
Analytical Science (Melbourne, Australia). This sorbent has particles with an average size
of 45 µm and nominal 60 Å porosity. Before using for the first time, it was conditioned
using 100 μL of methanol and then with 100 μL of water. The volumes of methanol and
water were drawn up and then discarded every time at an approximate flow rate of 20 μLs-1
(±5 μL s-1
)
The plasma and urine samples (50 μL each) were drawn through the syringe ten
times manually. It is important that samples are drawn slowly (approximately 20±5 μL s-1
)
and with caution to obtain good percolation between sample and solid support. The sorbent
was then washed once with 100 μL of water to remove proteins and other interferences.
The analytes were then eluted with 30 μL of methanol directly into the LC and GC
injector. The multiple pulling/pushing of the sample by the syringe increases the extraction
174
recovery. Between sample extractions, the C18 adsorbent in the barrel insert and needle
assembly was washed with methanol (4 × 50 μL) and water (4 × 50 μL). This step
decreased memory effects and also functioned as a conditioning step before the next
extraction. The same packing bed was used for about 100 extractions before it was
discarded.
4.2.5. Method Validation
Calibration curves of urine and plasma samples spiked with antidepressants standards were
performed in the range 1-500 ng mL-1
on GC-MS and 5-500 ng mL-1
on LC-UV with seven
concentration levels. The calibration curves were described by the linear regression
equation:
y = mx + c
where y is the peak area, x is the concentration, m is the slope and c is the intercept. The
limit of detection (LOD) was set at the concentration when the signal/noise ratio was equal
to 3:1. The quality control (QC) samples were prepared with the concentration of 5, 100
and 250 ng mL-1
for antidepressants. The accuracy and precision were calculated for the
QC samples, both within and between days. The experiments were done six times during
six different days. The extraction recoveries of the drugs were calculated by comparing the
peak areas of extracted QC samples from plasma and urine to the peak areas of analyte
standard solutions.
4.3. Results and Discussion
4.3.1. MEPS method development
The method using microextraction in packed syringe was used with C18 (4 mg) as sorbent
material. The recoveries from spiked samples were compared to that of calibration curve of
175
pure standard solutions by calculating the peak areas from chromatograms. The factors
affecting the absolute recovery were investigated such as the time and velocity of sample
loading and volume of eluting solvents. In MEPS the sample can be drawn through the
needle into the syringe, up and down, once or several times. Fig. 4.2 shows the effect of
such procedure using C18 as sorbent. Sample recovery was increased as we increased the
number of extraction cycles up to ten but after that recovery was almost the same. In this
way, the extraction cycles were optimized for the sample preparation step. Volume of the
eluting solvent methanol was also verified as 30 μL. The extraction time was only about
1.5 min and the recovery was in the range of 66-99%. To avoid any carry over the syringe
was washed four times by methanol and four times by water after each run.
4.3.2. LC -UV Analysis
Under the selected chromatographic conditions, citalopram, mirtazapine,
imipramine, amitriptyline and clomipramine appeared at 4.11, 5.89, 7.17, 7.98 and 10.98
min., respectively. Calibration curves were linear over the concentration range of 5-500 ng
mL-1
. The correlation coefficient was in the range of 0.981-0.993. The absolute detection
limits (LODs) of these antidepressants were in the range of 0.133-0.337 ng mL-1
(Table
4.1).
Representative chromatograms of blank and sample spiked with a concentration of
5 ng mL-1
of each antidepressant drug extracted by MEPS in urine and plasma samples on
LC-UV has been shown in Fig. 4.3 and Fig. 4.4. The extraction recoveries of all the drugs
were in the range of 66-98% in urine and plasma samples as shown in Table 4.2. The
RSDs at three different concentrations for quality control samples were less than 4.9 for
176
plasma samples (n=6) and lower than 5.3 for urine samples (n=6) on LC-UV. The accuracy
and the precision of the method were within the internationally accepted limits [42].
The carryover effect was investigated on the column by injecting into the liquid
chromatographic system three successive aliquots of a standard mixture containing all of
the analytes at a high concentration followed by three successive aliquots of extracted
blank urine and plasma. A non-significant carryover effect (less than 0.1%) was evident.
The method selectivity is defined as non-interference with the impurity substances in the
regions of interest. The developed method is very selective as MEPS-LC-UV analysis of
the blank urine sample and blank plasma sample showed no interfering peaks of impurity
compounds in the quantification of these antidepressant drugs.
4.3.3. GC-MS Analysis
A GC-MS method has been developed and optimized in order to determine the
extraction recovery of antidepressant drugs in spiked urine and blood samples. The
developed GC-MS method was optimized for: column temperature program, flow rate of
carrier gas, temperature of injector, ion source and interface. The final optimized GC
separation of analytes of interest was achieved within 18 min and the total
chromatographic run time was of 30.33 min. Mass spectra of these drugs are shown in Fig.
4.5 in both scan mode (4.5 a) and SIM mode (4.5 b). Due to higher specificity of GC-MS,
a compound can be easily recognized by its molecular ion and any other interference from
co-existing compounds can be identified, even if they co-elute.
Mass fragmentation of these drugs has been presented in Fig. 4.6. For detection in
SIM mode, the following ions were selected from their main fragmentation pattern
according to their abundance:
177
Amitriptyline: 58, 202, 277.
Imipramine: 58, 72, 136, 280.
Clomipramine: 58, 72,110, 242, 314.
Citalopram: 58, 262, 280, 324.
Mirtazapine: 72, 195, 265.
(*) Most abundant ion; (underlined) molecular ion.
The procedure yielded excellent separation and symmetrical peaks for each
antidepressant. Representative chromatograms of blanks and samples spiked with a
concentration of 5 ng mL-1
of each antidepressant drug extracted by MEPS in urine and
blood samples has been presented in Fig. 4.7 and Fig. 4.8. Under the described
chromatographic conditions, retention times were 15.5 min (amitriptyline), 15.8 min
(imipramine), 15.9 min (mirtazapine), 17.1 min (citalopram), and 17.3min (clomipramine).
The complete elution was obtained in less than 18 min.
Good linearity was obtained for all analytes with correlation coefficients
R2>0.981over a range of 1-500 ng mL
-1. Limits of detection (LOD) were calculated based
on signal-to noise (S/N =3) from spiked samples at low concentrations. LODs of these
antidepressants were ranged between 0.088-0.202 ng mL-1
. Limit of quantifications
(LOQs) (S/N = 10) for the analytes studied was in the range of 0.268-0.608 ng mL-1
(Table
4.1)
Table 4.3 shows the average recoveries of antidepressant drugs from spiked urine
and plasma samples at three different levels (5, 100 and 250 ng mL-1
) were in the range of
77-99 % in the urine and blood samples with relative standard deviations (RSDs) lower
than 6.2 %. The carry over was found to be less than 0.1%.
178
When urine and plasma samples spiked with a mixture of analytes was analysed on
GC-MS and compared with blank urine and blank plasma, no interfering compounds were
detected at the same retention times as of the studied compounds. Fig. 4.7 (A) and Fig. 4.8
(A) show good selectivity for MEPS as a sample preparation method.
4.4. Conclusions
A new, sensitive and accurate sample preparation technique MEPS was developed
and validated for the determination of antidepressants in the plasma and urine samples.
Compared with SPE, the new technique is more selective, robust, cost effective, fast and
fully automated. Also, small sample volumes can be treated (in μL) as well as large
volumes. The new technique can be used for complex matrices (such as blood) without any
problem. In MEPS, the packed sorbent can be used several times (100-200, depending on
the kind of matrix), whereas a conventional SPE cartridge is usually discarded after a
single use. This approach to sample analysis with LC-UV and GC-MS has been used
successfully in the quantitative analysis of citalopram, mirtazapine, imipramine,
amitriptyline and clomipramine in the urine and plasma samples. Although, GC-MS and
LC-UV are of comparable accuracy and precision, LC-UV has the advantages of very short
time of analysis. But as compared to LC-UV, GC-MS has higher sensitivity, selectivity
and feasibility of direct injection of samples into MS detector. Finally, unlike LC-UV, GC-
MS is capable of determining mixtures of antidepressants, even when they were not eluted
completely.
179
0
0.5
1
1.5
2
0 5 10 15 20 25
No. of cycles
mA
U
Citalopram
Imipramine
Mirtazapine
Amitriptyline
Clomipramine
Fig. 4.2 Effect of number of extraction cycles on extraction efficiency
180
0
0.5
1
1.5
2
0 2 4 6 8 10 12 14
Retention Time (min)
mA
U
Fig. 4.3 (A) MEPS/LC chromatograms obtained from blank urine
0
0.5
1
1.5
2
0 2 4 6 8 10 12 14
Retention Time (min)
mA
U
a
b
cd
e
Fig. 4.3 (B) MEPS/LC chromatograms obtained from spiked urine with Citalopram
(a), Mirtazapine (b), Imipramine (c), Amitriptyline (d), Clomipramine (e) at a
concentration of 5 ng mL-1
181
0
0.5
1
1.5
2
0 2 4 6 8 10 12 14
Retention Time (min)
mA
U
Fig. 4.4 (A) MEPS/LC chromatograms obtained from blank plasma
0
0.5
1
1.5
2
0 2 4 6 8 10 12 14
Retention Time (min)
mA
U
a
b
c
d
e
.
Fig. 4.4. (B) MEPS/LC chromatograms obtained from spiked plasma with
Citalopram (a), Mirtazapine (b), Imipramine (c), Amitriptyline (d), Clomipramine
(e) at a concentration of 5 ng mL-1
182
50 100 150 200 250 300 350 400 450 500 550 6000.0
25.0
50.0
75.0
100.0
%
84
105
51
207
267
136
355147 297195 251 401 443461356 484 570527 599548
Fig. 4.5 (A) Mass spectra of antidepressant drugs in scan mode
m/z
Rel
ativ
e In
tensi
ty
183
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.00.0
25.0
50.0
75.0
100.0
%
58
85269
195
280238202 314
Fig. 4.5 (B) Mass spectra of antidepressant drugs in SIM mode
m/z
Rel
ativ
e In
tensi
ty
184
O
N
F
N
CH3
CH3
Citalopram (325)
O
N
F
CH3+
N
N
N
CH3
N
F
CH2
+
m/z =280
m/z = 262
Mirtazapine (265)
N
N
CH3
CH3
Cl
Clomipramine (314)
44
58
72
86 242N
N
CH3
CH3
72
208
58
44
Imipramine (280)
N
CH3
CH3
Amitriptyline (277)
58
195
70
236
72
Fig. 4.6 Proposed fragmentation pattern for the antidepressant drugs observed
on GC-MS
185
0
5000
10000
15000
20000
25000
10 12 14 16 18 20
Retention Time (min)
Ab
un
dan
ce
Fig. 4.7 (A) MEPS/GC-MS chromatograms obtained from blank urine
0
5000
10000
15000
20000
25000
10 12 14 16 18 20
Retention Time (min)
Ab
un
dan
ce
d
c b a
e
Fig. 4.7 (B) MEPS/ GC-MS chromatograms obtained from spiked urine with
Citalopram (a), Mirtazapine (b), Imipramine (c), Amitriptyline (d), Clomipramine
(e) at a concentration of 5 ng mL-1
186
0
5000
10000
15000
20000
25000
10 12 14 16 18 20
Retention Time (min)
Ab
un
dan
ce
Fig. 4.8 (A) MEPS/ GC-MS chromatograms obtained from blank plasma
0
5000
10000
15000
20000
25000
10 12 14 16 18 20
Retention Time (min)
Ab
un
dan
ce
d
c abe
Fig. 4.8 (B) MEPS/ GC-MS chromatograms obtained from spiked plasma with
Citalopram (a), Mirtazapine (c), Imipramine (b), Amitriptyline (d), Clomipramine
(e) at a concentration of 5 ng mL-1
187
Table 4.1 LC-UV and GC-MS characteristics of antidepressant standards in water
Parameter
Amitriptyline Imipramine Mirtazapine Citalopram Clomipramine
HPLC-UV GC-MS HPLC-UV GC-MS HPLC-UV GC-MS HPLC-UV GC-MS HPLC-UV GC-MS
R2 0.9875 0.9892 0.9899 0.9978 0.9815 0.9940 0.9857 0.9976 0.9935
0.9874
Y 0.0438x
+4.7822
1231.7x-
45050
0.0293x
+5.0101
569.93x -
25145
0.0184x
+1.4789
592.77x -
21313
0.0315x
+5.1343
648.58x -
30803
0.0198x
+2.612
508.37x-
22156
LOD = 3.3 S/N
(ng mL-1
)
0.133 0.112 0.147 0.112 0.307 0.119 0.205 0.088 0.337 0.202
LOQ =10 S/N
(ng mL-1
) 0.390 0.330 0.432 0.340 0.930 0.354 0.618 0.268 0.986 0.608
RSD (%) 2.9 2.7 3.2 3.7 2.7 2.2 2.1 2.4 1.7 2.2
188
Table 4.2 Results for the determination of extraction yield of antidepressant drugs from spiked urine and plasma samples on LC-UV
a Each value is the mean of 6 independent assays. The extraction yield was calculated from analyte peak area from spiked urine
and plasma samples compared to those obtained from the same analyte concentration in standard solutions.
Analyte Amount added
(ng mL-1
)
Extraction yield (%)a Repeatability (RSD%)
a
Urine Plasma Urine Plasma
Amitriptyline 5.0
100
250
87.69
89.98
95.23
86.46
90.28
93.64
4.2
3.8
3.4
4.6
4.2
3.2
Imipramine 5.0
100
250
95.34
96.35
97.21
66.18
79.83
82.24
5.2
4.9
3.7
4.8
4.1
3.5
Mirtazapine 5
100
250
89.92
90.21
88.76
95.29
92.29
97.28
4.9
4.3
2.9
3.4
2.9
2.7
Citalopram 5.0
100
250
97.56
94.23
96.98
69.75
82.56
89.27
3.6
2.9
2.1
4.5
4.1
3.7
Clomipramine 5.0
100
250
70.08
91.29
84.25
90.12
92.25
94.41
3.1
2.5
1.9
3.9
4.1
3.5
189
Table 4.3 Results for the determination of extraction yield of antidepressant drugs from spiked urine and plasma samples on GC-MS
a Each value is the mean of 6 independent assays. The extraction yield was calculated from analyte peak area from spiked urine
and plasma samples compared to those obtained from the same analyte concentration in standard solutions.
Analyte Amount added
(ng mL-1
)
Extraction yield (%)a Repeatability (RSD%)
a
Urine Plasma Urine Plasma
Amitriptyline 5.0
100
250
97.73
98.93
97.92
81.47
92.85
95.54
3.5
3.1
2.7
4.2
3.7
3.2
Imipramine 5.0
100
250
97.52
96.54
98.27
81.33
94.97
92.21
6.1
5.2
4.8
5.9
4.8
3.9
Mirtazapine 5.0
100
250
97.19
95.42
99.10
81.02
95.58
93.14
5.4
4.1
3.6
4.4
2.6
1.9
Citalopram 5.0
100
250
95.84
97.28
94.36
79.86
89.58
91.64
3.8
2.9
2.7
4.9
3.6
2.5
Clomipramine 5.0
100
250
95.99
98.86
97.18
77.71
84.89
90.06
5.9
4.5
5.2
4.9
4.7
3.8
190
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