Abstract
In collaboration with the US Army, Alturas Analytics has developed several bioanalytical HPLC/MS/MS methods for the analysis of drugs used to protect against chemical warfare nerve agents.  Here we present the low-level GLP validation of an HPLC/MS/MS method for the determination of pyridostigmine bromide (PB) from guinea pig plasma.  In addition, we will discuss the development of an HPLC/MS/MS method for the determination of atropine from monkey serum.

PB is a pretreatment drug used to protect against chemical warfare nerve agents.  By itself PB is not protective against chemical warfare nerve agent poisoning.  However, when used as a pretreatment, PB might enhance the antidote effects of the standard atropine treatments used by the U.S. military for nerve agent poisoning.  Atropine is used immediately upon exposure to nerve gas agents to counteract the effects of the cholinesterase inhibitors.

Since the US Army continues to improve formulations, the low level quantitative analysis of these drugs in biological fluids is important.  For PB, the guinea pig PK experiments required the use of less than 30 uL of plasma with quantitation limits near 500 pg/mL.  For atropine, the monkey studies required limits of quantitation (LLOQ) near 1.0 pg/mL.

Here we report on the development of separate HPLC/MS/MS assays for the determination of PB from guinea pig plasma and atropine from monkey serum.  To accelerate sample analysis, all sample preparation was done in 96 well plates.  Deuterated internal standards were used for both assays.  High-speed gradient HPLC pumps were used in tandem with a Sciex API3000.  Multiple reaction monitoring was used to detect the drugs and internal standards.  The LLOQ for the atropine assay was less than 5.0 pg/mL whereas the LLOQ for the PB assay was 100 pg/mL using only 25 uL of plasma.  The run time for each assay was less than 2.0 minutes per sample.  This presentation illustrates a rapid, selective and sensitive method for the determination of PB and atropine in guinea pig and monkey serum, respectively.

Background
Shane Needham is Laboratory Director of Alturas Analytics, Inc where he manages the development and validation of HPLC/MS/MS assays to support the pharmaceutical industry.  He has more than 12 years of experience in analytical laboratories and has over 10 years of LC/MS and analytical lab experience in the drug discovery through drug development stage in the pharmaceutical industry.  Shane previously worked for Pfizer Central Research in Groton, CT where he developed and validated LC/MS/MS methods for the trace analysis of drugs and metabolites in biological fluids.  He also performed in vivo and in vitro structure elucidation studies of drugs and metabolites using HPLC/MS/MS.  Shane’s Ph.D. work included the development of optimized stationary phases to improve the assay of pharmaceuticals by HPLC/MS.  Shane has over 60 publications and external presentations in the area of LC/MS and teaches LC/MS courses on the national and international level.  Shane is a member of the American Chemical Society, Sigma Xi and American Society for Mass Spectrometry and American Association of Pharmaceutical Scientists.

Meeting details

Date:	Wednesday	March 26, 2003
Time:	6:00 pm	Social hour, registration (no-host cocktails)
	7:00 pm	Dinner
	8:00 pm	Lecture
Dinner:	Choice of:	Chicken Picatta with Roasted Red Potatoes
		Flank Steak with Chimi Churi Sauce
		Wild Rice and Mushroom Pilaf
	includes	(various side dishes & dessert)
Cost:	$25.00	BAMS members.  Reservations required by noon on Friday March 21, 2003
	$35.00	Non-members.  Reservations required by noon on Friday March 21, 2003
	$15.00	Students only.  Reservations required by noon on Friday March 21, 2003

Note: 2003 dues need to be paid to obtain member price.  Dues ($20) may be paid while registering for dinner.

Maps & directions
Roche Bioscience Cafeteria and Art Gallery Lecture Hall
3401 Hillview Ave
Palo Alto, CA 94304
(650) 855-5050
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The San Francisco Bay Area Mass Spectrometry discussion group was formed in 1980 to provide a regular gathering for people interested in mass spectrometry and allied topics. BAMS currently has a membership of about 280 individual and 20 corporate members, and meets 8-10 times per year for a midweek dinner and lecture.  Meetings attract between 30 and 90 people, and are held at a restaurant or hotel in the bay area convenient for our speaker.  We usually convene at 6:00 pm for cocktails, dinner at 7:00 pm, and lecture at 8:15 pm.

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Last update: 3/11/2003