Navigation Links
Determination of Acepromazine and its Major Metabolite in Equine Serum by LC-MS/MS using the Finnigan LCQ Deca XP Plus Ion Trap Mass Spectrometer

Chromatography and Mass Spectrometry Application Report

Key Words Sensitivity Quantitation Finnigan LCQ Deca XP Plus Equine serum Acepromazine

Gargi Choudhary, Thermo Electron Corporation, San Jose, CA USA
Wayne Skinner and Scott Stanley, University of California, Davis

Introduction

Acepromazine is widely used as a sedative in horses. It serves as a tranquilizer to better facilitate handling of these large mammals before transportation or surgical procedures. Acepromazine has been classified by the Association of Racing Commissioners International, Inc. as a Class 3 drug in horses. Abuse of the drugs in the Class 3 category can result in 2-6 months of suspension, up to $1500 fine, and loss of purse. Acepromazine has a slow elimination rate and stays in the body for a long time. This can be problematic if the drug is administered before a race event since its calming effect can lead to a better performance by the horse.

Goal

Develop a method for identifying and quantitating acepromazine (ACE) and its major metabolite 2-(1-hydroxyethyl) promazine sulfoxide (HEPS) in horse serum.
Determine limits of detection (LOD) and quantitation (LOQ) of these two compounds in serum.
Demonstrate low level detection of acepromazine in a complex biological matrix.

Experimental

HPLC
HPLC system: Surveyor MS pump with Surveyor autosampler
Column: 50 2.1 mm Hypersil BDS C18 (Thermo Electron)
Injection volume: 20 μL
Flow rate: 300 μL/min
Mobile phase A: Water containing 10 mM ammonium acetate and 0.1% formic acid
B: Acetonitrile containing 0.1% formic acid
Gradient: 5-40% B in 4 min, 40-65% B in 1 min, 65-90% B in 0.5 min, 90-5%B in 1 min, at 5% B for 4 min.

Mass Spectrometer
Mass spectrometer: Finnigan LCQ Deca XP Plus
Ionization mode: Positive ESI
Temperature: 300 C
Spray voltage: 4.5 kV
Sheath gas: 45 units
Sweep gas: 6 units
Isolation width: 1.5

Standards
Calibration standards were prepared as follows:

Samples
Acepromazine was administered to the horse, and serum samples were drawn 30, 45 min and 1, 2, 4, 6 and 24 hrs after dosing.

Internal Standard
Chlorpromazine at a concentration of 100 ng/mL (9:1 ACN:1 M acetic acid) was used as the internal standard.

Sample Preparation
0.5 mL of the calibration standard/horse serum sample was mixed with 0.6 mL of chlorpromazine internal standard. The standard/sample was then refrigerated for 30 min, centrifuged and the supernatant used for analysis. The resulting internal standard concentration in the calibration standard and the sample was 120 ng/mL of serum.

Results and Discussions
Full Scan LC-MS/MS Analysis

Figure 1 shows extracted ion chromatograms from fullscan MS/MS analysis of acepromazine (ACE), its major metabolite 2-(1-hydroxyethyl) promazine sulfoxide (HEPS) and the internal standard chlorpromazine. These chromatograms represent injection of horse serum containing 2 ng/mL of ACE and HEPS (40 pg on-column). The MS/MS spectra of the three analytes is also shown in Figure 1. The extracted ion chromatograms are generated by summing the intensities of the three most abundant ions in the MS/MS spectra as shown in Table 1.

Quantitation and Linear Dynamic Rang e Figures 2 and 3 show calibration curves for ACE and HEPS in horse serum with linearity over four orders of magnitude, i.e., 0.2 200 ng/mL. The R2 value is 0.9952 for ACE and 0.9922 for HEPS. These calibration curves were generated with chlorpromazine (120 ng/mL) as the internal standard and illustrate that the wide linear dynamic range of the Finnigan LCQ Deca XP Plus ion trap mass spectrometer enables quantitation of analytes in biological matrices.

Limit of Detection and Quantitation Figure 4 shows the extracted ion chromatograms for ACE and HEPS at a concentration of 0.5 ng/mL of serum. The signal to noise for ACE at 0.5 ng/mL of serum is 3:1 and hence represents the lower limit of detection (LOD) for this compound. The signal to noise for HEPS at concentration of 0.5 ng/mL of horse serum is 7:1 and represents the lower limit of quantitation (LOQ) for HEPS. Table 2 shows the lower limits of detection and quantitation for ACE and HEPS indicating that these analytes can be detected at low levels in horse serum with minimal sample preparation.

Determination of ACE and HEPS in Samples from the Horse
Figure 5 shows extracted ion chromatograms for ACE and HEPS obtained by full-scan MS/MS analysis of serum sample obtained 4-hr post administration of drug. No significant level of ACE could be detected in the horse at this time whereas its metabolite HEPS could be quantitated and its concentration determined as 1.3 ng/mL of serum. Determination of concentrations of ACE and HEPS in ng/mL of horse serum drawn at different time points post administration is tabulated in Table 3. It is seen that ACE could be detected in horse serum for up to 2 hr after dosing whereas its metabolite was detected for up to 6 hr.

Conclusions
Full-scan MS/MS analysis with a Finnigan LCQ Deca XP Plus ion trap mass spectrometer provides the selectivity and sensitivity necessary to support ADME studies of acepromazine in horse serum. Acepromazine was detected in horse serum for up to 2 hr after dosing whereas its metabolite was detected for up to 6 hr. The LOD achieved for acepromazine in horse serum was determined to be 0.5 ng/mL, while the LOQ for its metabolite was 0.5 ng/mL.

Acknowledgments
The authors would like to thank Lester Taylor and Diane Cho for their comments.


'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Structural Determination of Flavonoids Using MSn
2. Total Cellular Protein Determination Using the DC Protein Assay
3. Simultaneous Determination of Residues of Approximately 100 Pesticides and Metabolites in Fruit and Vegetables by LC/MS/MS
4. Determination of Polar Organophosphorus Pesticides in Aqueous Samples by Direct Injection Using HPLC/MS/MS
5. Determination of N-nitrosamines in Baby Bottle Rubber Teats by Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry
6. Determination of the Chloramphenicol residues in milk and milk products using LC/MS/MS
7. A Microtiter-based Assay for the Determination of ID50s of b-lactamase Inhibitors Employing Reporter Substrates Detected at UV or Visible Wavelengths (MaxLine Application Note #20)
8. Analysis of Nitrofurans Metabolites by Positive Ion Electrospray LC/MS/MS
9. Simultaneous Detection of CYP3A4, CYP2D6 and CYP2C9 Metabolites with a Single, Sensitive, LC/MS/MS Method
10. Identification of Phase I and Phase II Metabolites of Buspirone on the Q TRAP LC/MS/MS System
11. Strategies to Identify and Confirm Phase I Metabolites of Glyburide Using the 4000 Q TRAP System
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... ... 2016 , ... Parallel 6 , the leading software as a service ... Virtual Patient Encounter CONSULT module which enables both audio and video telemedicine communication ... , Using the CONSULT module, patients and physicians can schedule a face to face ...
(Date:6/27/2016)... PHILADELPHIA , June 27, 2016  Liquid ... today announced the funding of a Sponsored Research ... study circulating tumor cells (CTCs) from cancer patients.  ... changes in CTC levels correlate with clinical outcomes ... therapies. These data will then be employed to ...
(Date:6/24/2016)... DIEGO , June 24, 2016 ... more sensitively detects cancers susceptible to PARP inhibitors ... circulating tumor cells (CTCs). The new test has ... HRD-targeted therapeutics in multiple cancer types. ... targeting DNA damage response pathways, including PARP, ATM, ...
(Date:6/23/2016)... ... June 23, 2016 , ... Mosio, a leader in clinical ... Patient Recruitment and Retention Tips.” Partnering with experienced clinical research professionals, Mosio revisits ... tips, tools, and strategies for clinical researchers. , “The landscape of how patients ...
Breaking Biology Technology:
(Date:6/3/2016)... 3, 2016 Das ... Nepal hat ein 44 ... geprägter Kennzeichen, einschließlich Personalisierung, Registrierung und IT-Infrastruktur, ... Produktion und Implementierung von Identitätsmanagementlösungen. Zahlreiche renommierte ... Januar teilgenommen, aber Decatur wurde als konformste ...
(Date:5/24/2016)... superior patient care by providing unparalleled technology to leaders of the medical imaging industry. ... recently added to the range of products distributed by Ampronix. Photo - ... ... ... ...
(Date:5/3/2016)... 2016  Neurotechnology, a provider of high-precision biometric ... Biometric Identification System (ABIS) , a complete system ... ABIS can process multiple complex biometric transactions with ... fingerprint, face or iris biometrics. It leverages the ... MegaMatcher Accelerator , which have been used ...
Breaking Biology News(10 mins):