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Compound MRM Transition
Cone
Voltage (V)
Collision
Energy (eV)
Acetylcholine 146.10 > 87.05 25 12
d4-Acetylcholine 150.20 > 91.30 30 15
Choline 104.20 > 60.10 30 15
Histamine 112.20 > 95.12 25 15
d4-Histamine 116.10 > 99.00 25 15
tele-Methylhistamine 126.10 > 109.20 25 12
d3-tele-Methylhistamine 128.80 > 112.20 25 12
tele-Methylimidazole acetic acid 141.09 > 95.05 25 12
d3 tele-Methylimidazole acetic acid 144.10 > 98.10 25 12
DEVELOPMENT OF A QUANTITATIVE UPLC-MS/MS ASSAY FOR THE SIMULTANEOUS QUANTITATION OF ACETYLCHOLINE, HISTAMINE AND THEIR METABOLITES IN HUMAN CEREBRO-SPINAL FLUID (CSF)
Mary E. Lame, Erin E. Chambers, and Kenneth J. Fountain
Waters Corporation 34 Maple Street Milford MA, 01757 USA
References
1. Jia, J.P., et al., Differential acetylcholine and choline concentrations in
the cerebrospinal fluid of patients with Alzheimer's disease and vascular
dementia. Chin Med J (Engl), 2004. 117(8): p. 1161-4.
2. Ito, C., The role of the central histaminergic system on schizophrenia.
Drug News Perspect, 2004. 17(6): p. 383-7.
3. Prell, G.D. and J.P. Green, Measurement of histamine metabolites in
brain and cerebrospinal fluid provides insights into histaminergic activity.
Agents Actions, 1994. 41 Spec No: p. C5-8.
4. Huang, T., et al., Detection of basal acetylcholine in rat brain
microdialysate. J Chromatogr B Biomed Appl, 1995. 670(2): p. 323-7.
5. Bourgogne, E., et al., Simultaneous quantitation of histamine and its
major metabolite 1-methylhistamine in brain dialysates by using
precolumn derivatization prior to HILIC-MS/MS analysis. Anal Bioanal
Chem, 2012. 402(1): p. 449-59.
6. Zhang, Y., et al., Development and validation of a sample stabilization
strategy and a UPLC-MS/MS method for the simultaneous quantitation of
acetylcholine (ACh), histamine (HA), and its metabolites in rat
cerebrospinal fluid (CSF). J Chromatogr B Analyt Technol Biomed Life Sci,
2011. 879(22): p. 2023-33.
CONCLUSIONS
A fast, simple, sensitive and selective analytical scale UPLC
method was developed for separation and simultaneous
quantitation of ACh, HA and their metabolites in human CSF.
The 96-well format allowed for sample preparation in <15
minutes, providing the high throughput required for
discovery and development analysis.
Resolution and sensitivity of the CORTECS UPLC HILIC
column improved separation and resolution from endogenous matrix components and allowed for analysis
times of 2.5 minutes.
The high sensitivity of the Xevo™ TQ-S MS system
facilitated the use of small sample volumes (20 L) and a
5X dilution to achieve detection limits as low as 10 pg/mL
with a broad quantitative dynamic range of 10 pg/mL-30,000 pg/mL.
QC samples for all analytes easily passed FDA regulatory
criteria, with % CV’s of 0.3-10.1%, and an accuracy range
of 96.0-111.1 % for all analytes tested, indicating a very reproducible and accurate method.
The method described herein, shows promise for highly
selective and sensitive quantification of multiple neurological biomarkers in the discovery and development
stages of pharmaceutical drug development.
INTRODUCTION
Biochemical biomarkers, derived from bodily fluids, are
often used in drug discovery and development as a
useful way of identifying disease or effectiveness of drug treatment. Acetylcholine (ACh) and Histamine (HA) are
highly polar neurotransmitters that act in the peripheral and central nervous system. They play a role in sleep
regulation, memory and learning, and immune responses. A decrease in ACh levels in the brain is a
known contributor in memory dysfunction and is in particularly short supply in people with Alzheimer's
disease[1]. It has been shown that metabolites of histamine are increased in the cerebrospinal fluid (CSF)
of people with schizophrenia [2]. Given the physiological importance of both ACh, HA and their metabolites, the
ability to measure small changes in their physiological concentrations as a function of disease progression or
drug treatment make them highly advantageous as
biochemical biomarkers.
The development of targeted assays for ACh, HA and their respective metabolites, is a persistent challenge due
to the demand for high sensitivity, selectivity and the need for fast sample analysis times. Additionally,
simultaneous measurement requires a broad quantitative dynamic range due to the vast difference in endogenous
circulating concentrations of ACh, HA, and their metabolites. While there are a number of analytical
methodologies for quantifying these analytes (GC/MS, HPLC-EC, and LC/MS))[3-5] there are few in which ACh,
HA and their metabolites have been quantified simultaneously [6]. Hydrophilic interaction liquid chroma-
tography (HILIC) is increasingly becoming the technique
of choice for these challenging polar analyte separations due to improved retention of polar species, orthogonal
selectivity to reversed-phase chromatography for mix-tures of polar and ionizable compounds, and increased
mass spectrometry response.
The method described herein demonstrates the simulta-neous quantification of ACh, HA, and their metabolites
(Figure 1) in human CSF in a 96-well format. This appli-cation uses a single step sample preparation, dilution of
a 20 µL sample, followed by HILIC UPLC-MS/MS analysis. The use of a 1.6 µm, high efficiency CORTECS™ HILIC
UPLC® column provided resolution from endogenous ma-trices, increased analyte sensitivity, and a short analysis
time of 2.5 minutes, resulting in a fast, sensitive, selec-
tive, and accurate method that meets the demands of high throughput bioanalytical drug discovery.
Figure 1: Representative structures of ACh, HA, and their
respective metabolites.
Acetylcholine (ACh) Choline (Ch)
Histamine (HA) tele-methylhistamine (t-mHA) tele-methyimidazoleacetic acid (t-MIAA)
METHODS
SAMPLE PREPARATION
20 µL of artificial CSF (aCSF) standard, aCSF quality control (QC), or human CSF (containing 4 mM eSerine)
QC samples were transferred to a 1 mL 96-well plate. 100 µL of acetonitrile containing 1 ng/mL each of d4-
Acetylcholine, d4-Histamine, d3-tele-Methylhistamine, and d3-tele-Methylimidazolacetic, which were used as
internal standards (ISTD), was added to the samples.
The plate was covered and vortexed. The samples were then centrifuged for 5 minutes at 4000 rpm and 10 µL of
sample was analyzed by UPLC-MS/MS.
ACQUITY UPLC CONDITIONS
Column: CORTECS UPLC HILIC
2.1 x 100 mm, 1.6 µm Mobile Phase A: 100 mM Ammonium Formate, pH 3 in
H2O Mobile Phase B: Acetonitrile
Flow Rate: 0.50 mL/min Gradient: Time Profile Curve
(min) %A %B 0.0 10 90 6
0.01 10 90 6 0.75 40 60 6
1.00 40 60 6 1.25 70 30 6
1.90 70 30 6 1.91 10 90 6
Injection Volume: 10.0 µL Column Temperature: 45 °C
Sample Temperature: 6 °C
Waters Xevo™ TQ-S CONDITIONS, ESI+ Capillary Voltage 3.0 kV Desolvation Temp 550 °C
Cone gas Flow 150 L/hr Desolvation Gas Flow 900 L/hr
Collision Cell Pressure 3.58 X 10 (-3) mbar Collision Energy and cone voltage: Optimized by
component, see Table 1
Table 2. Analytical performance of the UPLC-MS/MS assay for ACH, Ch,
Compound Dynamic Range (pg/mL) Linearity (1/x)
LLQC
(pg/mL)
Mean QC
Accuracy
Acetylcholine 10-10,000 0.998 16 102.2
Choline 100-30,000 0.999 140 103.3
Histamine 50-10,000 0.998 62 105.1
tele-Methylhistamine 10-10,000 0.999 16 102.2
tele-Methylimidazole acetic acid 20-30000 0.999 35 97.6
LLQC: Lower Limit of Quantification Control
ACh
HA
t-MIAA
t-mHA
iso-AChCh
Figure 2. UPLC-MS/MS analysis of ACh, Ch, iso-ACh, HA, t-mHA,
and t-MIAA in aCSF. All peak widths, at base, are between 2.1 and 3.7 seconds wide.
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
MRM of 10 Channels ES+ 126.1 > 109.2 (t-MHA)
3.43e4
MRM of 10 Channels ES+ 112.2 > 95.12 (HA)
5.39e4
MRM of 10 Channels ES+ 141.1 > 95.17 (MIAA)
1.14e5
MRM of 10 Channels ES+ 104.2 > 60.1 (CH)
1.30e5
MRM of 10 Channels ES+ 146.1 > 87.05 (ACH)
6.69e4
1.73
1.61
1.53
1.45
1.36 ACh
HA
t-MIAA
t-mHA
Ch
Figure 3. Representative chromatograms of ACh, Ch, HA, t-mHA, and t-
MIAA in aCSF at LLQC concentrations (16 pg/mL for Ach/t-mHA, 62pg/mL for HA, 35 pg/mL for t-MIAA, and 140 pg/mL for Ch)
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0
100
MRM of 10 Channels ES+ 146.1 > 87.05 (ACH)
2.06e7
ACh
iso-ACh
1.36
1.58
Figure 4. Representative chromatogram of ACh and iso-ACH in Human
CSF (ACh Overspike of 1000 pg/mL) demonstrating the chromatographic separation and concentration differences between ACh and iso-ACh.
Table 3. Representative ACh, HA, and t-mHA results from the
analysis of QC samples prepared in human CSF.
Table 5. Summary of measured mean endogenous basal levels in hu-
man CSF for ACh, Ch, HA, t-mHA, and t-MIAA, respectively.
RESULTS/DISCUSSION
A particular challenge in development of this assay was
maintaining high sensitivity while chromatographically
resolving the analytes of interest from both mobile phase and endogenous matrix interferences. The method
developed was optimized with buffer strength and careful attention to gradient profile to balance the elution profile for
optimal peak shape, resolution, and run time without sacrificing sensitivity or selectivity for ACh, HA and their
metabolites. Chromatographic retention and performance of ACh, Ch, and their respective metabolites is shown in Figure
2. ACh, HA, their metabolites and an isobar of ACh, iso-ACh (3-carboxypropyl)trimethylammonium), eluted between
1.35 and 1.75 minutes, with peak widths less than 3.7 seconds at base. Representative chromatograms for the
lower limit of quantification control (LLQC) samples of ACh, HA and their metabolites in aCSF are shown in Figure 3.
Figure 4, an extracted human CSF sample, highlights the
resolution of ACh from the high endogenous concentration of iso-ACh present.
The broad dynamic range and ability to distinguish small
changes of these neurotransmitters as a result of disease progression or drug treatment with high accuracy and
reproducibly is critical. Dynamic ranges, linearity, average QC accuracy, and lower limit of quantitation control (LLOQ)
are shown in Table 2. A representative standard curve for ACh is shown in Figure 5. Average accuracy and precision
values for QC samples were 96.0-111.1, with CV ranges of 0.3-10.1% (Tables 3 and 4), indicating a reproucible and
accurate method easily meeting the FDA regulatory criteria for LC-MS/MS assays. Following qualification of aCSF and
human CSF calibrators, the endogenous basal
concentrations of each analyte in human CSF were determined using the aCSF standard curves. A summary of
all mean determined basal levels of ACh, Ch, HA, t-mHA, and t-MIAA is listed in Table 5.
The combination of a simple sample preparation with
analysis using a highly selective UPLC HILIC method, based on a sub-2µm CORTECS HILIC UPLC column, and a sensitive
MS system easily provided the required sensitivity, selectivity, and resolution from endogenous matrix
components. The above mentioned benefits also facilitated short analysis times of 2.5 minutes and the use of sample
volumes of 20 L to achieve detection limits as low as 10
pg/mL, with a broad quantitative dynamic range of 10 pg/
mL-30,000 pg/. This method shows promise for its use in
accurately quantifying multiple biomarkers in the discovery and development stages of pharmaceutical drug
development.
Table 1. MRM transitions, collision energies, and cone voltages for
ACh, Ch, HA, t-mHA, t-MIAA, and their respective deuterated stable isotope-labeled internal standards (ISTDs).
Figure 5. Representative standard curve for ACh prepared in aCSF.
Table 4. Representative t-MIAA and Ch results from the
analysis of QC samples prepared in aCSF.