1
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation 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 XevoTQ-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 H 2 O 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-ACh Ch 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. Time 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 % 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) Time 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 % 0 100 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.

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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation

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.