Assurance Scientific Laboratories, 727 Memorial Dr, Suite 103, Bessemer AL ITSP Solutions Inc., 10 South Carolina St., Hartwell GA 30643

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1 Unified Drug Testing by Online SPE-LC/MS/MS Focus on Productivity Achieved Through Ease of Use: One totally automated method measures ALL the drugs in urine and/or oral fluids Mark Hayward, 2 Rick Youngblood, 2 Kim Gamble, 2 Jonathan Ho, 3 Tom Moran, 3 Martin Johnson, 1 and Matthew T. Hardison 1 1 Assurance Scientific Laboratories, 727 Memorial Dr, Suite 103, Bessemer AL ITSP Solutions Inc., 10 South Carolina St., Hartwell GA Syringe 3 Shimadzu Scientific Instruments 19 Schoolhouse Rd, Suite 107, Someset NJ SPE cartridge Positive pressure micro scale SPE Automated like this!

2 Measurement of drugs of abuse in urine and/or oral fluids (OF) is common Pre-employment screening DOT / federal mandated testing Law enforcement Compliance / diagnostic determinations by physicians Latter two are growing rapidly! Fastest growing & preferred approach is LC/MS/MS Due to high degree of certainty for simultaneous identification and concentration determination Continued growth in the use of LC/MS/MS for the measurement of drugs of abuse in urine and OF seems certain

3 Still, there are several technical challenges that need to be met Easily measure low dose drugs at/near 1 ng/g For medical purposes [Pesce, et. al AACC conference] as well as for zero tolerance testing Simplicity for performing measurements with lab technicians Automation: load samples / run list (prioritized queuing for STAT samples) All LC/MS/MS peaks sufficiently intense for automatic integration Ability to achieve high productivity for all work Needs apply to ALL testing! [not just high volume tests] Investing 90% effort in 10% of tests is not acceptable Minimizing the labor and number of workflows required Simplicity has always been crucial As volume and number of tests continues to grow, simplicity grows in importance!

4 Pain Management

5 Perhaps the increase in the use of pain meds is, in part, the price we pay for increasing longevity through medicine

6 Pain Management Drug Monitoring Required to prevent abuse, addiction, diversion, mortality and morbidity (urine drug testing) Nevertheless, must meet patient needs first (and not penalize low, irregular dosing) Production environment: assembly line Needs to be easy, robust, and have low labor requirements to measure all relevant drugs at all relevant concentrations Needs sufficient capacity relative to capital investment: 100 reimbursable reports per day per LC/MS/MS How does this impact ones approach toward the measurement methodology?

7 Measurement range (defining the challenge): low single digit ng/g for some opioids and benzos Pesce, et. al AACC conference Dilute and shoot is insufficient Measurement at these levels usually requires some pre-concentration of the sample SPE, LLE At the same time, this needs to be easy! automation

8 Approach chosen: SPE performed with LC/MS/MS autosampler Automated (serial) pre-concentration of samples, so that all drugs can be measured in one method Remove salts, proteins, and cells for robust LC/MS/MS operation Very little high LogP(greasy) interferences Primary role is to filter, pre-concentrate, and remove salts / other H 2 O soluble interferences Reverse phase should be sufficient Modest capital investment: must buy LC autosamplerregardless choose one that does more of the work CTC/PAL ITSP ITSP = Instrument Top Sample Prep

9 What is ITSP? Micro-SPE performed by a CTC/PAL ITSP = Instrument Top Sample Prep Photos: Assurance Scientific Laboratories Precise syringe driven flow ITSP SPE: overall system, AKA your autosampler! ITSP SPE cartridge being discarded after use ITSP SPE cartridge mg sorbent 10 mg most common and has 32 µl internal volume

10 Comparison of SPE technologies Technology Capabilities SPE Technologies ITSP syringe flow SPE Parallel tube and plate based SPE Pipette based SPE (packed sorbent) Pipette based SPE (loose sorbent, dispersive) HPLC Pump flow SPE (2D HPLC) Total automation Y Y-but separate workflow flow from LC/MS and GC/MS [extra robots = extra $$$] Y-but separate workflow flow from LC/MS and GC/MS [extra robots = extra $$$] Y-but separate workflow flow from LC/MS and GC/MS [extra robots = extra $$$] Y-requires additional pumps, valves, and fixed columns [extra LC hardware = extra $$$] On-line operation w/ LC/MS/MS Y N N N Y On-line operation w/ GC/MS/MS Y N N N N Accurate Flow Control Y N N N Y Single use sorbent Y Y Y Y N All sorbents available Y Y N N N Pre-concentrate w/o dry down Y N N N Y Fast Y N Y N Y Method development automation Y N N N N Off-line operation to feed multiple instruments Y Y Y Y N Full chain of custody Works with any MS software Ease of use & maintenance Pros Cons Y-bar code reading possible at every step Y-SW pre-embedded in all major MS brands Y-the PAL is a HPLC/GC autosampler& requires same skills & maintenance Automation, accuracy, & multiple tests on single system set up None Y -with manual labeling and recording of each step N N N N -separate work flow N -separate work flow N -separate work flow Conceptually simple, but laborious and flow control is challenging. Robots similar to pipette based SPE. Many methods in literature Laborious and lack of accurate flow control Easy to do once one has bought, set up, and learned how to use separate robotic pipette systems ($$$) Easy to do once one has bought, set up, and learned how to use separate robotic pipette systems ($$$) Limited sorbent choices and bed masses. Flow control not proven. Easy to do once one has bought, set up, and learned how to use separate robotic pipette systems ($$$) Easy to do once one has bought, set up, and learned how to use separate robotic pipette systems ($$$) Not proven to improve sample condition and no control of flow over sorbent Y -each MS manufacturer has some unique form of 2D HPLC N Single workflow automation Requires significant expertise and one never knows when/how SPE column will fail (limits applications)

11 Understanding ITSP and how it differs from other approaches ITSP has precise flow control allowing separations to be performed at their Van Deemter optimum velocity This yields separation performance that is difficult (if not impossible) to achieve with other SPE approaches ITSP also is a completely unique form of SPE in that it is a truly low volume device (32 µl) This allows elution to performed precisely with µl volumes (ca. 50 µl ready for direct injection on LC/MS or GC/MS) This allows ITSP to easily pre-concentrate samples by loading 1-10 ml of sample while eluting with <100 µl of solvent Other forms of SPE require larger volumes for elution (10 to 50x) and significant pre-concentration can only be achieved by adding a separate slow, laborious dry-down step No other SPE approach can achieve the precise chromatographic separations, pre-concentration of sample, robust operation, and total automation achieved simultaneously by ITSP

12 ITSP = TOTAL CONTRO OL OF YOUR SPE! % Rec covery SPE is Chromatography! Optimized outcomes require accurate flow Are you optimized? Or just guessing? Oxycodone Van Deemtercurves for SPE by ITSP 90 SCX %-Recovery in 20% Water SCX %-Recovery in 80% Water Reverse Phase %-Recovery RP optimum velocity = 1.5 mm/s (4x higher than SCX!) Flow (µl/s) [5 µl/s = 1.5 mm/s] SCX optimum velocity = 0.37 mm/s (1.2 µl/s by ITSP) with little room for error! SPE flow driven pneumatically or by vacuum cannot achieve and maintain optimal flow! ITSP = TOTAL CO ONTROL OF YOUR SPE!

13 SPE-LC/MS/MS method development helicopter view of strategy Focus on simplicity and minimization of steps Prioritize hardest to measure drugs (lowest concentration, low dose opioidsand benzos) over the easy to measure drugs for recovery optimization and pre-concentration Focus on relative recoveries for elution pre-concentrate Choose balanced LC/MS conditions that allow separation and measurement of both acidic and basic drugs as well as polar and non-polar drugs (1 method, all drugs!) Establish linear scalability and stoichiometryin sample loading as a data driven way to establish the validation readiness of the method Leverage automation to achieve rapid method development and execution ITSP with the CTC/PAL Develop the method for urine first, then adapt to oral fluid

14 Serially automated SPE method development Parallel testing of C 18 and DVB SPE phases (3x): each step is a sample list! Hands on view of strategy Test SPE cartridge wash with various solvents (3x cartridge volume) and no wash: rinse cartridge with water, load spiked urine & measure drug breakthroughs (choose wash solvent) Test SPE cartridge conditioning & loaded sample wash with buffers (at 3x cartridge volume): load spiked urine & measure drug breakthroughs (choose conditioning/wash buffer) Test SPE cartridge elution with various solvents and measure drug recoveries (choose elution solvent and measure optimal flow) Test SPE cartridge elution at multiple volumes with various buffers in chosen elution solvents and measure drug recoveries (pre-concentrate) while monitoring LC separation (choose elution buffer based on LC separation first, then recoveries) Vary sample amount over a range of at least 10x and measure drug recoveries. If linear stoichiometryis not observed, re-optimize above steps based on data. If linear, re-optimize LC/MS/MS, choose sample amount, then validate!

15 Automated method development Run each of the 5 lists sequentially (described in previous slide) transferring optima measured into next list Optima results: Sorbent: C 18 end-capped (gives higher recovery for benzos/ opioids) Conditioning: MeOH, then NH 4 OAc buffer Minimizes Loading: 0.5% NH 4 OAc in sample, Flow 5 µl/s breakthrough Wash: aqueous NH 4 OAc ( 0.5%) Elution: 0.2% NH 4 OAc in MeOH, 75 µl at 5 µl/s (preconcentrates lowest concentration drugs most) Sample load range: µl is linear at 0.5% NH 4 OAc in sample (can be increased with higher %NH 4 OAc in sample)

16 Test compatibility of SPE eluentwith LC separation (SPE LC interfacing) Of course, chemical presentation of the sample from SPE to LC is important Hydrocodone SPE eluent Buffered 80% ACN 80% ACN Just like with SPE, control of the ph (ionization state) controls retention Codeine Buffer: NH 4 OAc LC column: C 18 B = ACN Elution in 80% ACN limits LC injection volume to 2 µl (2.1 x 50 mm column). Elution in 100% MeOH(buffered) allows 5 µl LC injection. Viscosity has an equally important role in LC injection along with ph.

17 SPE elution volume Allows optimization for drug classes 1 C 18 SPE data shown with MeOH elution DVB with MeOHelution favors low volume elution for all drugs Normalized res sponse k = k = 2-3 k > 3 THCA 6-MAM Buprenorphine Codeine Diazepam Secobarbital Phencyclidine 0.4 Favors opiates, metabolites, and other illicits Favors opioids, benzos, barbs, and THCA Best for PM Elution volume (ul) Dilutes all drugs Gains in sensitivity from lower volume elution using DVB do not outweigh the absolute recoveries observed with C 18 SPE

18 Sample loading: defining SPE capacity and linear working range Linear response observed within 100 to 500 µl sample load range for all PM drugs Current triple quads (TQs) can measure all PM drugs in the lower half of this range Opiates, metabolites, other illicitssaturate buffer capacity (not cartridge) first at 500 µl sample load Performance below 100 µl can be improved with smaller syringe and blowing out cartridge with air at each step ( RTC) [also dilution to 200 µl with PAL works nicely] ITSP cartridge volume is 32 µl Normalized LC/MS/MS res sponse ROI optimum Pre-concentration 1.5-3x Current TQs Older TQs Linear range Opiates, metabolites, & illicitsfully functional (recoveries 90%) Opioidsand benzosare most optimized by design (recoveries >95%) Volume (µl) of urine loaded on SPE cartridge C 18 SPE, MeOHelution, 0.5% NH 4 OAc in sample Linear range can be extended with higher %NH 4 OAc in sample MDMA Fentanyl Amphetamine Diazepam Oxymorphone Pentobarbital THCA 500 µl syringe loading sample at 5 µl/s

19 Focus on the LC/MS/MS also can be productive Original LC/MS/MS It works 4.5 min Heat column, increase flow, add gradient segments Improve separation where peaks are crowded Decrease time between well separated peaks 4.1 min Column switching / conditioning also saves overhead time

20 Acidic drugs can be measured under LC conditions used for basic drugs THCA Pentobarbital Secobarbital

21 Workflow: minimizing cycle time PAL operation in the inject ahead mode SPE 1 SPE 2 SPE 3 LC/MS/MS 1 LC/MS/MS min 4.5 min Total cycle time (SPE + LC/MS/MS) = 4.5 min

22 Summary: Drugs in urine An online and automated SPE-LC/MS/MS method has been developed for pain management (PM) monitoring in urine samples The method is scalable and can be adapted to any LC/MS/MS simply by adjusting the volume of urine loaded for SPE Rather than using multiple panel focused methods, this single methodis used to measure all PM related drugs Method development focus has been on the lowest dose, hardest to measure prescribed drugs (suggests prioritization works!) Rework is limited to only the highest dose drugs in the highest dosed patients (inject less, bring into linear range) Robust operation and a cycle time (SPE + LC/MS/MS) of 4.5 min has been achieved The use of ITSP with the CTC/PAL for serial automation is a very efficient way to perform SPE method development: this method required 3 lab days using 1 SPE-LC/MS/MS system

23 Adapt method developed for urine to function equally well for oral fluid (OF) First things first: choosing a sampling device Many considered / criticized, primary concerns: Sample stability Volumetric sampling accuracy We added need for forensicacceptance and success when challenged at trial Quantisal sampling device chosen for its: Highest volumetric accuracy (+/-10%) Built in filtering (important: all OF samples require either filtering or centrifugation) >1 week sample stability Greatest acceptance in clinical and forensic use

24 Adapt / test method for oral fluid (OF) SPE and sample buffering increased to 2% NH4OAc to overcome built in Quantisalbuffering High opiate / illicit drug recovery requires formation of drug-oac ion pair Sample volume increased to 1 ml to achieve 0.1 ng/g cut offs Drug concentrations lower in OF 13x pre-concentration Linear response for 71 drugs achieved Method tested with 20 multi medication / not so good prognosis patient samples where both urine and OF collected in parallel and tested with both LC/MS/MS and immunoassay LC/MS/MS in complete agreement for urine and OF (no false neg) and partying successfully detected in one sample set Included patients dosed 1 mg/day opioids/ benzos:easily measured / auto-integrated at approx 0.5 ng/g in OF (0.1 ng/g cutoffs) Immunoassay showed one of its deficiencies by not being able to detect / confirm 1 mg/day dosed drugs (urine and OF)

25 Unifiedurine and oral fluid method results Lab time to adapt urine method to OF: 2 days Again, leveraging the automation Method (urine & OF) validated to both clinical and forensic standards in multiple labs (cv 3-5%) Urine and OF can be measured in the same run Still need to include blanks, calibrators, and QCs for both Method (urine & OF) in use for production work and delivering results overnight for each LC/MS/MS High ROI readily achieved for top end LC/MS/MSs

26 What s next? Larger range of tests ready to run with a single LC/MS/MS We have successfully added VitD (blood) measurement to individual PAL systems already measuring drugs in urine / OF These 3 tests are currently considered highest ROI in clinical lab 2 cartridge types, 2 LC column types, all on-board, all the time We have drug methods for blood/serum/plasma/dps/dbs samples to add to these systems (ion exchange SPE) Includes DPS/DBS disk extraction and IS addition We are implementing SPE solvent sourcing to Fast Wash stations (up to 4) as an alternative to ink wells Up to 8 solvents total using minimal PAL rail Allows 2-4 weeks of solvent capacity (0.5-2 liter bottles) We are implementing4 way valve cleaning solvent sourcing for DLW (multiple cleaning chemistries for multiple methods) This should allow 4 tests to be setup/ready to run on single PAL-LC/MS/MS systems requiring only method selection in MS software and perhaps loading color coded SPE cartridges

27 Smart SPE Acknowledgement Peter Ratsep Scott Cassidy